Nucleic acid encoding delta-9 desaturase

ABSTRACT

The present invention relates to nucleic acid molecules encoding delta 9 desaturase gene, and expression vectors, plant cells, and transgenic plants expressing delta 9 desaturase nucleic acid. The nucleic acid molecules of the present invention can be used, for example, to decrease delta 9 desaturase activity in plant cells, resulting in decreased unsaturated fatty acid production.

This application is a continuation-in-part of: 1) a Non-Provisionalapplication by Edington, entitled “Method for the production oftransgenic plants deficient in starch granule bound glucose starchglycosyl transferase activity” filed on Sep. 2, 1994 as U.S. Ser. No. of08/300,726; and 2) a Provisional application by Zwick et al., entitled“Composition and method for modification of fatty acid saturationprofile in plants” filed on Jul. 13, 1995, as U.S. Ser. No. 60/001,135.Both of these applications in their entirety, including drawings, arehereby incorporated by reference herein.

BACKGROUND OF THE INVENTION

The present invention concerns compositions and methods for themodulation of gene expression in plants, specifically using enzymaticnucleic acid molecules.

The following is a brief description of regulation of gene expression inplants. The discussion is not meant to be complete and is provided onlyfor understanding of the invention that follows. This summary is not anadmission that any of the work described below is prior art to theclaimed invention.

There are a variety of strategies for modulating gene expression inplants. Traditionally, antisense RNA (reviewed in Bourque, 1995 PlantSci 105, 125-149) and co-suppression (reviewed in Jorgensen, 1995Science 268, 686-691) approaches have been used to modulate geneexpression. Insertion mutagenesis of genes have also been used tosilence gene expression. This approach, however, cannot be designed tospecifically inactivate the gene of interest. Applicant believes thatribozyme technology offers an attractive new means to alter geneexpression in plants.

Naturally occurring antisense RNA was first discovered in bacteria overa decade ago (Simons and Kleckner, 1983 Cell 34, 683-691). It is thoughtto be one way in which bacteria can regulate their gene expression(Green et al., 1986 Ann. Rev. Biochem. 55: 567-597; Simons 1988 Gene 72:35-44). The first demonstration of antisense-mediated inhibition of geneexpression was reported in mammalian cells (Izant and Weintraub 1984Cell 36: 1007-1015). There are many examples in the literature for theuse of antisense RNA to modulate gene expression in plants. Followingare a few examples:

Shewmaker et al., U.S. Pat. Nos. 5,107,065 and 5,453,566 disclosemethods for regulating gene expression in plants using antisense RNA.

It has been shown that an antisense gene expressed in plants can act asa dominant suppressor gene. Transgenic potato plants have been producedwhich express RNA antisense to potato or cassava granule bound starchsynthase (GBSS). In both of these cases, transgenic plants have beenconstructed which have reduced or no GBSS activity or protein. Thesetransgenic plants give rise to potatoes containing starch withdramatically reduced amylose levels (Visser et al. 1991, Mol. Gen.Genet. 225: 2889-296; Salehuzzaman et al. 1993, Plant Mol. Biol. 23:947-962).

Kull et al., 1995, J. Genet. & Breed. 49, 69-76 reported inhibition ofamylose biosynthesis in tubers from transgenic potato lines mediated bythe expression of antisense sequences of the gene for granule-boundstarch synthase (GBSS). The authors, however, indicated a failure to seeany in vivo activity of ribozymes targeted against the GBSS RNA.

Antisense RNA constructs targeted against Δ-9 desaturase enzyme incanola have been shown to increase the level of stearic acid (C18:0)from 2% to 40% (Knutzon et. al., 1992 Proc. Natl. Acad. Sci. 89, 2624).There was no decrease in total oil content or germination efficiency inone of the high stearate lines. Several recent reviews are availablewhich illustrate the utility of plants with modified oil composition(Ohlrogge, J. B. 1994 Plant Physiol. 104, 821; Kinney, A. J. 1994 Curr.Opin. Cell Biol. 5, 144; Gibson et al. 1994 Plant Cell Envir. 17, 627).

Homologous transgene inactivation was first documented in plants as anunexpected result of inserting a transgene in the sense orientation andfinding that both the gene and the transgene were down-regulated (Napoliet al., 1990 Plant Cell 2: 279-289). There appears to be at least twomechanisms for inactivation of homologous genetic sequences. One appearsto be transcriptional inactivation via methylation, where duplicated DNAregions signal endogenous mechanisms for gene silencing. This approachof gene modulation involves either the introduction of multiple copiesof transgenes or transformation of plants with transgenes with homologyto the gene of interest (Ronchi et al. 1995 EMBO J. 14: 5318-5328). Theother mechanism of co-suppression is post-transcriptional, where thecombined levels of expression from both the gene and the transgene isthought to produce high levels of transcript which triggersthreshold-induced degradation of both messages (van Bokland et al., 1994Plant J. 6: 861-877). The exact molecular basis for co-suppression isunknown.

Unfortunately, both antisense and co-suppression technologies aresubject to problems in heritability of the desired trait (Finnegan andMcElroy 1994 Bio/Technology 12: 883-888). Currently, there is no easyway to specifically inactivate a gene of interest at the DNA level inplants (Pazkowski et al., 1988 EMBO J. 7: 4021-4026). Transposonmutagenesis is inefficient and not a stable event, while chemicalmutagenesis is highly non-specific.

Applicant believes that ribozymes present an attractive alternative andbecause of their catalytic mechanism of action, have advantages overcompeting technologies. However, there have been difficulties indemonstrating the effectiveness of ribozymes in modulating geneexpression in plant systems (Mazzolini et al., 1992 Plant Mol. Biol. 20:715-731; Kull et al., 1995 J. Genet. & Breed. 49: 69-76). Although thereare reports in the literature of ribozyme activity in plants cells,almost all of them involve down regulation of exogenously introducedgenes, such as reporter genes in transient assays (Steinecke et al.,1992 EMBO J. 11:1525-1530; Perriman et al., 1993 Antisense Res. Dev. 3:253-263; Perriman et al., 1995, Proc. Natl. Acad. Sci. USA, 92, 6165).

There are also several publications, [e.g., Lamb and Hay, 1990, J. Gen.Virol. 71, 2257-2264; Gerlach et al., International PCT Publication No.WO 91/13994; Xu et al., 1992, Science in China (Ser. B) 35, 1434-1443;Edington and Nelson, 1992, in Gene Regulation: Biology of antisense RNAand DNA, eds. R. P. Erickson and J. G. Izant, pp 209-221, Raven Press,NY.; Atkins et al., International PCT Publication No. WO 94/00012; Leneeet al., International PCT Publication Nos. WO 94/19476 and WO 9503404,Atkins et al., 1995, J. Gen. Virol. 76, 1781-1790; Gruber et al., 1994,J. Cell. Biochem. Suppl. 18A, 110 (X1-406) and Feyter et al., 1996, Mol.Gen. Genet. 250, 329-338], that propose using hammerhead ribozymes tomodulate: virus replication, expression of viral genes and/or reportergenes. None of these publications report the use of ribozymes tomodulate the expression of plant genes.

Mazzolini et al., 1992, Plant. Mol. Bio. 20, 715-731; Steinecke et al.,1992, EMBO J. 11, 1525-1530; Perriman et al., 1995, Proc. Natl. Acad.Sci. USA., 92, 6175-6179; Wegener et al., 1994, Mol. Gen. Genet. 245,465-470; and Steinecke et al., 1994, Gene, 149, 47-54, describe the useof hammerhead ribozymes to inhibit expression of reporter genes in plantcells.

Bennett and Cullimore, 1992 Nucleic Acids Res. 20, 831-837 demonstratehammerhead ribozyme-mediated in vitro cleavage of glna, glnb, glng andglnd RNA, coding for glutamine synthetase enzyme in Phaseolus vulgaris.

Hitz et al., (WO 91/18985) describe a method for using the soybean Δ-9desaturasc enzyme to modify plant oil composition. The applicationdescribes the use of soybean Δ-9 desaturase sequence to isolate Δ-9desaturase genes from other species.

The references cited above are distinct from the presently claimedinvention since they do not disclose and/or contemplate the use ofribozymes in maize. Furthermore, Applicant believes that the referencesdo not disclose and/or enable the use of ribozymes to down regulategenes in plant cells, let alone plants.

SUMMARY OF THE INVENTION

The invention features modulation of gene expression in plantsspecifically using enzymatic nucleic acid molecules. Preferably, thegene is an endogenous gene. The enzymatic nucleic acid molecule with RNAcleaving activity may be in the form of, but not limited to, ahammerhead, hairpin, hepatitis delta virus, group I intron, group IIintron, RNaseP RNA, Neurospora VS RNA and the like. The enzymaticnucleic acid molecule with RNA cleaving activity may be encoded as amonomer or a multimer, preferably a multimer. The nucleic acids encodingfor the enzymatic nucleic acid molecule with RNA cleaving activity maybe operably linked to an open reading frame. Gene expression in anyplant species may be modified by transformation of the plant with thenucleic acid encoding the enzymatic nucleic acid molecules with RNAcleaving activity. There are also numerous technologies for transforminga plant: such technologies include but are not limited to transformationwith Agrobacterium, bombarding with DNA coated microprojectiles,whiskers, or electroporation. Any target gene may be modified with thenucleic acids encoding the enzymatic nucleic acid molecules with RNAcleaving activity. Two targets which are exemplified herein are delta 9desaturase and granule bound starch synthase (GBSS).

Until the discovery of the inventions herein, nucleic acid-basedreagents, such as enzymatic nucleic acids (ribozymes), had yet to bedemonstrated to modulate and/or inhibit gene expression in plants suchas monocot plants (e.g., corn). Ribozymes can be used to modulate aspecific trait of a plant cell, for example, by modulating the activityof an enzyme involved in a biochemical pathway. It may be desirable, insome instances, to decrease the level of expression of a particulargene, rather than shutting down expression completely: ribozymes can beused to achieve this. Enzymatic nucleic acid-based techniques weredeveloped herein to allow directed modulation of gene expression togenerate plant cells, plant tissues or plants with altered phenotype.

Ribozymes (i.e., enzymatic nucleic acids) are nucleic acid moleculeshaving an enzymatic activity which is able to repeatedly cleave otherseparate RNA molecules in a nucleotide base sequence-specific manner.Such enzymatic RNA molecules can be targeted to virtually any RNAtranscript, and efficient cleavage has been achieved in vitro and invivo (Zaug et al., 1986, Nature 324, 429; Kim et al., 1987, Proc. Natl.Acad. Sci. USA 84, 8788; Dreyfus, 1988, Einstein Quarterly J. Bio. Med.,6, 92; Haseloff and Gerlach, 1988, Nature 334 585; Cech, 1988, JAMA 260,3030; Murphy and Cech, 1989, Proc. Natl. Acad. Sci. USA., 86, 9218;Jefferies et al., 1989, Nucleic Acids Research 17, 1371).

Because of their sequence-specificity, trans-cleaving ribozymes may beused as efficient tools to modulate gene expression in a variety oforganisms including plants, animals and humans (Bennett et al., supra;Edington et al., supra; Usman & McSwiggen, 1995 Ann. Rep. Med. Chem. 30,285-294; Christoffersen and Marr, 1995 J. Med. Chem. 38, 2023-2037).Ribozymes can be designed to cleave specific RNA targets within thebackground of cellular RNA. Such a cleavage event renders the mRNAnon-functional and abrogates protein expression from that RNA. In thismanner, synthesis of a protein associated with a particular phenotypeand/or disease state can be selectively inhibited.

Other features and advantages of the invention will be apparent from thefollowing description of the preferred embodiments thereof, and from theclaims.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a diagrammatic representation of the hammerhead ribozymedomain known in the art. Stem II can be ≧2 base-pairs long. Each N isany nucleotide and each • represents a base pair.

FIG. 2a is a diagrammatic representation of the hammerhead ribozymedomain known in the art; FIG. 2b is a diagrammatic representation of thehammerhead ribozyme as divided by Uhlenbeck (1987, Nature, 327, 596-600)into a substrate and enzyme portion; FIG. 2c is a similar diagramshowing the hammerhead divided by Haseloff and Gerlach (1988, Nature,334, 585-591) into two portions; and FIG. 2d is a similar diagramshowing the hammerhead divided by Jeffries and Symons (1989, Nucl.Acids. Res., 17, 1371-1371) into two portions.

FIG. 3 is a diagrammatic representation of the general structure of ahairpin ribozyme. Helix 2 (H2) is provided with a least 4 base pairs(i.e., n is 1, 2, 3 or 4) and helix 5 can be optionally provided oflength 2 or more bases (preferably 3-20 bases, i.e., m is from 1-20 ormore). Helix 2 and helix 5 may be covalently linked by one or more bases(i.e., r is ≧1 base). Helix 1, 4 or 5 may also be extended by 2 or morebase pairs (e.g., 4-20 base pairs) to stabilize the ribozyme structure,and preferably is a protein binding site. In each instance, each N andN′ independently is any normal or modified base and each dash representsa potential base-pairing interaction. These nucleotides may be modifiedat the sugar, base or phosphate. Complete base-pairing is not requiredin the helices, but is preferred. Helix 1 and 4 can be of any size(i.e., o and p is each independently from 0 to any number, e.g., 20) aslong as some base-pairing is maintained. Essential bases are shown asspecific bases in the structure, but those in the art will recognizethat one or more may be modified chemically (abasic, base, sugar and/orphosphate modifications) or replaced with another base withoutsignificant effect. Helix 4 can be formed from two separate molecules,i.e., without a connecting loop. The connecting loop when present may bea ribonucleotide with or without modifications to its base, sugar orphosphate. “q” is ≧2 bases. The connecting loop can also be replacedwith a non-nucleotide linker molecule. H refers to bases A, U, or C. Yrefers to pyrimidine bases. “—” refers to a covalent bond.

FIG. 4 is a representation of the general structure of the hepatitis Δvirus ribozyme domain known in the art.

FIG. 5 is a representation of the general structure of the self-cleavingVS RNA ribozyme domain.

FIG. 6 is a schematic representation of an RNaseH accessibility assay.Specifically, the left side of FIG. 6 is a diagram of complementary DNAoligonucleotides bound to accessible sites on the target RNA.Complementary DNA oligonucleotides are represented by broad lineslabeled A, B, and C. Target RNA is represented by the thin, twistedline. The right side of FIG. 6 is a schematic of a gel separation ofuncut target RNA from a cleaved target RNA. Detection of target RNA isby autoradiography of body-labeled, T7 transcript. The bands common toeach lane represent uncleaved target RNA; the bands unique to each lanerepresent the cleaved products.

FIG. 7 is a graphical representation of RNaseH accessibility of GBSSRNA.

FIG. 8 is a graphical representation of GBSS RNA cleavage by ribozymesat different temperatures.

FIG. 9 is a graphical representation of GBSS RNA cleavage by multipleribozymes.

FIGS. 10A-C list the nucleotide sequence of Δ-9 desaturase cDNA isolatedfrom Zea mays.

FIGS. 11 and 12 are diagrammatic representations of fatty acidbiosynthesis in plants. FIG. 11 has been adapted from Gibson et al.,1994, Plant Cell Envir. 17, 627.

FIGS. 13 and 14 are graphical representations of RNaseH accessibility ofΔ-9 desaturase RNA.

FIG. 15 shows cleavage of Δ-9 desaturase RNA by ribozymes in vitro.10/10 represents the length of the binding arms of a hammerhead (HH)ribozyme. 10/10 means helix 1 and helix 3 each form 10 base-pairs withthe target RNA (FIG. 1). 4/6 and 6/6, represent helix2/helix1interaction between a hairpin ribozyme and its target. 4/6 means thehairpin (HP) ribozyme forms four base-paired helix 2 and a sixbase-paired helix 1 complex with the target (see FIG. 3). 6/6 means, thehairpin ribozyme forms a 6 base-paired helix 2 and a six base-pairedhelix 1 complex with the target. The cleavage reactions were carried outfor 120 min at 26° C.

FIG. 16 shows the effect of arm-length variation on the activity of HHand HP ribozymes in vitro. 7/7, 10/10 and 12/12 are essentially asdescribed above for the HH ribozyme. 6/6, 6/8, 6/12 represents varyinghelix 1 length and a constant (6 bp) helix 2 for a hairpin ribozyme. Thecleavage reactions were carried out essentially as described above.

FIGS. 17, 18, 19 and 23 are diagrammatic representations of non-limitingstrategies to construct a transcript comprising multiple ribozyme motifsthat are the same or different, targeting various sites within Δ-9desaturase RNA.

FIGS. 20 and 21 show in vitro cleavage of Δ-9 desaturase RNA byribozymes that are transcribed from DNA templates using bacteriophage T7RNA polymerase enzyme.

FIG. 22 diagrammatic representation of a non-limiting strategy toconstruct a transcript comprising multiple ribozyme motifs that arc thesame or different targeting various sites within GBSS RNA.

FIG. 24 shows cleavage of Δ-9 desaturase RNA by ribozymes. 453 Multimer,represents a multimer ribozyme construct targeted against hammerheadribozyme sites 453, 464, 475 and 484. 252 Multimer, represents amultimer ribozyme construct targeted against hammerhead ribozyme sites252, 271, 313 and 326. 238 Multimer, represents a multimer ribozymeconstruct targeted against three hammerhead ribozyme sites 252, 259 and271 and one hairpin ribozyme site 238 (HP). 259 Multimer, represents amultimer ribozyme construct targeted against two hammerhead ribozymesites 271 and 313 and one hairpin ribozyme site 259 (HP).

FIG. 25 illustrates GBSS mRNA levels in Ribozyme minus Controls (C, F,I, J, N, P, Q) and Active Ribozyme RPA63 Transformants (AA, DD, EE, FF,GG, HH, JJ, KK).

FIG. 26 illustrates Δ9 desaturase mRNA levels in Non-transformed plants(NT), 85-06 High Stearate Plants (1, 3, 5, 8, 12, 14), and Transformed(irrelevant ribozyme)

FIG. 27 illustrates Δ9 desaturase mRNA levels in Non-transformed plants(NTO), 85-15 High Stearate Plants (01, 06, 07, 10, 11, 12), and 85-15Normal Stearate Plants (02, 05, 09, 14).

FIG. 28 illustrates Δ9 desaturase mRNA levels in Non-transformed plants(NTY), 113-06 Inactive Ribozyme Plants (02, 04, 07, 10, 11).

FIGS. 29a and 29 b illustrate Δ9 desaturase protein levels in maizeleaves (R0). (a) Line HiII, plants a-e nontransformed and ribozymeinactive line RPA113-17, plants 1-6. (b) Ribozyme active line RPA85-5,plants 1-15.

FIG. 30 illustrates stearic acid in leaves of RPA85-06 plants.

FIG. 31 illustrates stearic acid in leaves of RPA85-15 plants, resultsof three assays.

FIG. 32 illustrates stearic acid in leaves of RPA113-06 plants.

FIG. 33 illustrates stearic acid in leaves of RPA113-17 plants.

FIG. 34 illustrates stearic acid in leaves of control plants.

FIG. 35 illustrates leaf stearate in R1 plants from a high stearateplant cross (RPA85-15.07 self).

FIG. 36 illustrates Δ9 desaturase levels in next generation maize leaves(R1). * indicates those plants that showed a high stearate content.

FIG. 37 illustrates stearic acid in individual somatic embryos from aculture (308/430-012) transformed with antisense Δ9 desaturase.

FIG. 38 illustrates stearic acid in individual somatic embryos from aculture (308/430-015) transformed with antisense Δ9 desaturase.

FIG. 39 illustrates stearic acid in individual leaves from plantsregenerated from a culture (308/430-012) transformed with antisense Δ9desaturase.

FIG. 40 illustrates amylose content in a single kernel of untransformedcontrol line (Q806 and antisense line 308/425-12.2.1.

FIG. 41 illustrates GBSS activity in single kernels of a southernnegative line (RPA63-0306) and Southern positive line RPA63-0218.

FIG. 42 illustrates a transformation vector that can be used to expressthe enzymatic nucleic acid of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention concerns compositions and methods for themodulation of gene expression in plants specifically using enzymaticnucleic acid molecules.

The Following Phrases and Terms are Defined Below

By “inhibit” or “modulate” is meant that the activity of enzymes such asGBSS and Δ-9 desaturase or level of mRNAs encoded by these genes isreduced below that observed in the absence of an enzymatic nucleic acidand preferably is below that level observed in the presence of aninactive RNA molecule able to bind to the same site on the mRNA, butunable to cleave that RNA.

By “enzymatic nucleic acid molecule” it is meant a nucleic acid moleculewhich has complementarity in a substrate binding region to a specifiedgene target, and also has an enzymatic activity which is active tospecifically cleave that target. That is, the enzymatic nucleic acidmolecule is able to internolecularly cleave RNA (or DNA) acid therebyinactivate a target RNA molecule. This complementarty functions to allowsufficient hybridization of the enzymatic nucleic acid molecule to thetarget RNA to allow the cleavage to occur. One hundred percentcomplementarity is preferred, but complementarity as low as 50-75% mayalso be useful in this invention. The nucleic acids may be modified atthe base, sugar, and/or phosphate groups. The term enzymatic nucleicacid is used interchangeably with phrases such as ribozymes, catalyticRNA, enzymatic RNA, catalytic DNA, nucleozyme, DNAzyme, RNA enzyme,RNAzyme, polyribozymes, molecular scissors, self-splicing RNA,self-cleaving RNA, cis-cleaving RNA, autolytic RNA, endoribonuclease,minizyme, leadzyme or DNA enzyme. All of these terminologies describenucleic acid molecules with enzymatic activity. The term encompassesenzymatic RNA molecule which include one or more ribonucleotides and mayinclude a majority of other types of nucleotides or abasic moieties, asdescribed below.

By “complementarity” is meant a nucleic acid that can form hydrogenbond(s) with other RNA sequences by either traditional Watson-Crick orother non-traditional types (for example, Hoogsteen type) of base-pairedinteractions.

By “vectors” is meant any nucleic acid- and/or viral-based techniqueused to deliver and/or express a desired nucleic acid.

By “gene” is meant a nucleic acid that encodes an RNA.

By “plant gene” is meant a gene encoded by a plant.

By “endogenous” gene is meant a gene normally found in a plant cell inits natural location in the genome.

By “foreign” or “heterologous” gene is meant a gene not normally foundin the host plant cell, but that is introduced by standard gene transfertechniques.

By “nucleic acid” is meant a molecule which can be single-stranded ordouble-stranded, composed of nucleotides containing a sugar, a phosphateand either a purine or pyrimidine base which may be same or different,and may be modified or unmodified.

By “genome” is meant genetic material contained in each cell of anorganism and/or a virus.

By “mRNA” is meant RNA that can be translated into protein by a cell.

By “cDNA” is meant DNA that is complementary to and derived from a mRNA.

By “dsDNA” is meant a double stranded cDNA.

By “sense” RNA is meant RNA transcript that comprises the mRNA sequence.

By “antisense RNA” is meant an RNA transcript that comprises sequencescomplementary to all or part of a target RNA and/or mRNA and that blocksthe expression of a target gene by interfering with the processing,transport and/or translation of its primary transcript and/or mRNA. Thecomplementarity may exist with any part of the target RNA, i.e., at the5′ non-coding sequence, 3′ non-coding sequence, introns, or the codingsequence. Antisense RNA is normally a mirror image of the sense RNA.

By “expression”, as used herein, is meant the transcription and stableaccumulation of the enzymatic nucleic acid molecules, mRNA and/or theantisense RNA inside a plant cell. Expression of genes involvestranscription of the gene and translation of the mRNA into precursor ormature proteins.

By “cosuppression” is meant the expression of a foreign gene, which hassubstantial homology to an gene, and in a plant cell causes thereduction in activity of the foreign and/or the endogenous proteinproduct.

By “altered levels” is meant the level of production of a gene productin a transgenic organism is different from that of a normal ornon-transgenic organism.

By “promoter” is meant nucleotide sequence element within a gene whichcontrols the expression of that gene. Promoter sequence provides therecognition for RNA polymerase and other transcription factors requiredfor efficient transcription. Promoters from a variety of sources can beused efficiently in plant cells to express ribozymes. For example,promoters of bacterial origin, such as the octopine synthetase promoter,the nopaline synthase promoter, the manopine synthetase promoter;promoters of viral origin, such as the cauliflower mosaic virus (35S);plant promoters, such as the ribulose-1,6-biphosphate (RUBP) carboxylasesmall subunit (ssu), the beta-conglycinin promoter, the phaseolinpromoter, the ADH promoter, heat-shock promoters, and tissue specificpromoters. Promoter may also contain certain enhancer sequence elementsthat may improve the transcription efficiency.

By “enhancer” is meant nucleotide sequence element which can stimulatepromoter activity (Adh).

By “constitutive promoter” is meant promoter element that directscontinuous gene expression in all cells types and at all times (actin,ubiquitin, CaMV 35S).

By “tissue-specific” promoter is meant promoter element responsible forgene expression in specific cell or tissue types, such as the leaves orseeds (zein, oleosin, napin, ACP).

By “development-specific” promoter is meant promoter element responsiblefor gene expression at specific plant developmental stage, such as inearly or late embryogenesis.

By “inducible promoter” is meant promoter element which is responsiblefor expression of genes in response to a specific signal, such as:physical stimulus (heat shock genes); light (RUBP carboxylase); hormone(Em); metabolites; and stress.

By a “plant” is meant a photosynthetic organism, either eukaryotic andprokaryotic.

By “angiosperm” is meant a plant having its seed enclosed in an ovary(e.g., coffee, tobacco, bean, pea).

By “gymnosperm” is meant a plant having its seed exposed and notenclosed in an ovary (e.g., pine, spruce).

By “monocotyledon” is meant a plant characterized by the presence ofonly one seed leaf (primary leaf of the embryo). For example, maize,wheat, rice and others.

By “dicotyledon” is meant a plant producing seeds with two cotyledons(primary leaf of the embryo). For example, coffee, canola, peas andothers.

By “transgenic plant” is meant a plant expressing a foreign gene.

By “open reading frame” is meant a nucleotide sequence, without introns,encoding an amino acid sequence, with a defined translation initiationand termination region.

The invention provides a method for producing a class of enzymaticcleaving agents which exhibit a high degree of specificity for the RNAof a desired target. The enzymatic nucleic acid molecule may be targetedto a highly specific sequence region of a target such that specific geneinhibition can be achieved. Alternatively, enzymatic nucleic acid can betargeted to a highly conserved region of a gene family to inhibit geneexpression of a family of related enzymes. The ribozymes can beexpressed in plants that have been transformed with vectors whichexpress the nucleic acid of the present invention.

The enzymatic nature of a ribozyme is advantageous over othertechnologies, since the concentration of ribozyme necessary to affect atherapeutic treatment is lower. This advantage reflects the ability ofthe ribozyme to act enzymatically. Thus, a single ribozyme molecule isable to cleave many molecules of target RNA. In addition, the ribozymeis a highly specific inhibitor, with the specificity of inhibitiondepending not only on the base-pairing mechanism of binding to thetarget RNA, but also on the mechanism of target RNA cleavage. Singlemismatches, or base-substitutions, near the site of cleavage cancompletely eliminate catalytic activity of a ribozyme.

Six basic varieties of naturally-occurring enzymatic RNAs are knownpresently. Each can catalyze the hydrolysis of RNA phosphodiester bondsin trans (and thus can cleave other RNA molecules) under physiologicalconditions. Table I summarizes some of the characteristics of theseribozymes. In general, enzymatic nucleic acids act by first binding to atarget RNA. Such binding occurs through the target binding portion of anenzymatic nucleic acid which is held in close proximity to an enzymaticportion of the molecule that acts to cleave the target RNA. Thus, theenzymatic nucleic acid first recognizes and then binds a target RNAthrough complementary base-pairing, and once bound to the correct site,acts enzymatically to cut the target RNA. Strategic cleavage of such atarget RNA will destroy its ability to direct synthesis of an encodedprotein. After an enzymatic nucleic acid has bound and cleaved its RNAtarget, it is released from that RNA to search for another target andcan repeatedly bind and cleave new targets.

In one of the preferred embodiments of the inventions herein, theenzymatic nucleic acid molecule is formed in a hammerhead or hairpinmotif, but may also be formed in the motif of a hepatitis Δ virus, groupI intron, group II intron or RNaseP RNA (in association with an RNAguide sequence) or Neurospora VS RNA. Examples of such hammerhead motifsare described by Dreyfus, supra, Rossi et al., 1992, AIDS Research andHuman Retroviruses 8, 183; of hairpin motifs by Hampel et al.,EP0360257, Hampel and Tritz, 1989 Biochemistry 28, 4929, Feldstein etal., 1989, Gene 82, 53, Haseloff and Gerlach, 1989, Gene, 82, 43, andHampel et al., 1990 Nucleic Acids Res. 18, 299; of the hepatitis Δ virusmotif is described by Perrotta and Been, 1992 Biochemistry 31, 16; ofthe RNaseP motif by Guerrier-Takada et al., 1983 Cell 35, 849; Forsterand Altman, 1990, Science 249, 783; Li and Altman, 1996, Nucleic AcidsRes. 24, 835; Neurospora VS RNA ribozyme motif is described by Collins(Saville and Collins, 1990 Cell 61, 685-696; Saville and Collins, 1991Proc. Natl. Acad. Sci. USA 88, 8826-8830; Collins and Olive, 1993Biochemistry 32, 2795-2799; Guo and Collins, 1995, EMBO. J. 14, 363);Group II introns are described by Griffin et al., 1995, Chem. Biol. 2,761; Michels and Pyle, 1995, Biochemistry 34, 2965; and of the Group Iintron by Cech et al., U.S. Pat. No. 4,987,071. These specific motifsare not limiting in the invention and those skilled in the art willrecognize that all that is important in an enzymatic nucleic acidmolecule of this invention is that it has a specific substrate bindingsite which is complementary to one or more of the target gene RNAregions, and that it have nucleotide sequences within or surroundingthat substrate binding site which impart an RNA cleaving activity to themolecule.

The enzymatic nucleic acid molecules of the instant invention will beexpressed within cells from eukaryotic promoters [e.g., Gerlach et al.,International PCT Publication No. WO 91/13994; Edington and Nelson,1992, in Gene Regulation: Biology of Antisense RNA and DNA, eds. R. P.Erickson and J. G. Izant, pp 209-221, Raven Press, NY.; Atkins et al.,International PCT Publication No. WO 94/00012; Lenee et al.,International PCT Publication Nos. WO 94/19476 and WO 9503404, Atkins etal., 1995, J. Gen. Virol. 76, 1781-1790; McElroy and Brettell, 1994,TIBTECH 12, 62; Gruber et al., 1994, J. Cell. Biochem. Suppl. 18A, 110(X1-406)and Feyter et al., 1996, Mol. Gen. Genet. 250, 329-338; all ofthese are incorporated by reference herein]. Those skilled in the artwill realize from the teachings herein that any ribozyme can beexpressed in eukaryotic plant cells from an appropriate promoter. Theribozymes expression is under the control of a constitutive promoter, atissue-specific promoter or an inducible promoter.

To obtain the ribozyme mediated modulation, the ribozyme RNA isintroduced into the plant. Although examples are provided below for theconstruction of the plasmids used in the transformation experimentsillustrated herein, it is well within the skill of an artisan to designnumerous different types of plasmids which can be used in thetransformation of plants, see Bevan, 1984, Nucl. Acids Res. 12,8711-8721, which is incorporated by reference. There are also numerousways to transform plants. In the examples below embryogenic maizecultures were helium blasted. In addition to using the gene gun (U.S.Pat. No. 4,945,050 to Cornell and U.S. Pat. No. 5,141,131 to DowElanco),plants may be transformed using Agrobacterium technology, sec U.S. Pat.No. 5,177,010 to University of Toledo, U.S. Pat. No. 5,104,310 to TexasA&M, European Patent Application 0131624B1, European Patent Applications120516, 159418B1 and 176,112 to Schilperoot, U.S. Pat. Nos. 5,149,645,5,469,976, 5,464,763 and 4,940,838 and 4,693,976 to Schilperoot,European Patent Applications 116718, 290799, 320500 all to MaxPlanck,European Patent Applications 604662 and 627752 to Japan Tobacco,European Patent Applications 0267159, and 0292435 and U.S. Pat. No.5,231,019 all to Ciba Geigy, U.S. Pat. Nos. 5,463,174 and 4,762,785 bothto Calgene, and U.S. Pat. Nos. 5,004,863 and 5,159,135 both toAgracetus; whiskers technology, see U.S. Pat. Nos. 5,302,523 and5,464,765 both to Zeneca; electroporation technology, see WO 87/06614 toBoyce Thompson Institute, U.S. Pat. No. 5,472,869 and U.S. Pat. No.5,384,253 both to Dekalb, WO9209696 and WO9321335 both to PGS; all ofwhich are incorporated by reference herein in totality. In addition tonumerous technologies for transforming plants, the type of tissue whichis contacted with the foreign material (typically plasmids containingRNA or DNA) may vary as well. Such tissue would include but would not belimited to embryogenic tissue, callus tissue type I and II, and anytissue which is receptive to transformation and subsequent regenerationinto a transgenic plant. Another variable is the choice of a selectablemarker. The preference for a particular marker is at the discretion ofthe artisan, but any of the following selectable markers may be usedalong with any other gene not listed herein which could function as aselectable marker. Such selectable markers include but are not limitedto chlorosulfuron, hygromyacin, PAT and/or bar, bromoxynil, kanamycinand the like. The bar gene may be isolated from Strptomuces,particularly from the hygroscopicus or viridochromogenes species. Thebar gene codes for phosphinothricin acetyl transferase (PAT) thatinactivates the active ingredient in the herbicide bialaphosphosphinothricin (PPT). Thus, numerous combinations of technologies maybe used in employing ribozyme mediated modulation.

The ribozymes may be expressed individually as monomers, i.e., oneribozyme targeted against one site is expressed per transcript.Alternatively, two or more ribozymes targeted against more than onetarget site are expressed as part of a single RNA transcript. A singleRNA transcript comprising more than one ribozyme targeted against morethan one cleavage site are readily generated to achieve efficientmodulation of gene expression. Ribozymes within these multimerconstructs are the same or different. For example, the multimerconstruct may comprise a plurality of hammerhead ribozymes or hairpinribozymes or other ribozyme motifs. Alternatively, the multimerconstruct may be designed to include a plurality of different ribozymemotifs, such as hammerhead and hairpin ribozymes. More specifically,multimer ribozyme constructs arc designed, wherein a series of ribozymemotifs are linked together in tandem in a single RNA transcript. Theribozymes are linked to each other by nucleotide linker sequence,wherein the linker sequence may or may not be complementary to thetarget RNA. Multimer ribozyme constructs (polyribozymes) are likely toimprove the effectiveness of ribozyme-mediated modulation of geneexpression.

The activity of ribozymes can also be augmented by their release fromthe primary transcript by a second ribozyme (Draper et al., PCT WO93/23569, and Sullivan et al., PCT WO 94/02595, both hereby incorporatedin their totality by reference herein; Ohkawa, J., et al., 1992, NucleicAcids Symp. Ser., 27, 15-6; Taira, K., et al., 1991, Nucleic Acids Res.,19, 5125-30; Ventura, M., et al., 1993, Nucleic Acids Res., 21, 3249-55;Chowrira et al., 1994 J. Biol. Chem. 269, 25856).

Ribozyme-mediated modulation of gene expression can be practiced in awide variety of plants including angiosperms, gymnosperms,monocotyledons, and dicotyledons. Plants of interest include but are notlimited to: cereals, such as rice, wheat, barley, maize; oil-producingcrops, such as soybean, canola, sunflower, cotton, maize, cocoa,safflower, oil palm, coconut palm, flax, castor, peanut; plantationcrops, such as coffee and tea; fruits, such as pineapple, papaya, mango,banana, grapes, oranges, apples; vegetables, such as cauliflower,cabbage, melon, green pepper, tomatoes, carrots, lettuce, celery,potatoes, broccoli; legumes, such as soybean, beans, peas; flowers, suchas carnations, chrysanthemum, daisy, tulip, gypsophila, alstromeria,marigold, petunia, rose; trees such as olive, cork, poplar, pine; nuts,such as walnut, pistachio, and others. Following are a few non-limitingexamples that describe the general utility of ribozymes in modulation ofgene expression.

Ribozyme-mediated down regulation of the expression of genes involved incaffeine synthesis can be used to significantly change caffeineconcentration in coffee beans. Expression of genes, such as7-methylxanthosine and/or 3-methyl transferase in coffee plants can bereadily modulated using ribozymes to decrease caffeine synthesis (Adamsand Zarowitz, U.S. Pat. No. 5,334,529; incorporated by referenceherein).

Transgenic tobacco plants expressing ribozymes targeted against genesinvolved in nicotine production, such as N-methylputrescine oxidase orputrescine N-methyl transferase (Shewmaker et al., supra), would produceleaves with altered nicotine concentration.

Transgenic plants expressing ribozymes targeted against genes involvedin ripening of fruits, such as ethylene-forming enzyme, pectinmethyltransferase, pectin esterase, polygalacturonase,1-amininocyclopropane carboxylic acid (ACC) synthase, ACC oxidase genes(Smith et al., 1988, Nature, 334, 724; Gray et al., 1992, Pl. Mol.Biol., 19, 69; Tieman et al., 1992, Plant Cell, 4, 667; Picton et al.,1993, The Plant J. 3, 469; Shewmaker et al., supra; James et al., 1996,Bio/Technology, 14, 56), would delay the ripening of fruits, such astomato and apple.

Transgenic plants expressing ribozymes targeted against genes involvedin flower pigmentation, such as chalcone synthase (CHS), chalconeflavanone isomerase (CHI), phenylalanine ammonia lyase, ordehydroflavonol (DF) hydroxylases, DF reductase (Krol van der, et al.,1988, Nature, 333, 866; Krol van der, et al., 1990, Pl. Mol. Biol., 14,457; Shewmaker et al., supra; Jorgensen, 1996, Science, 268, 686), wouldproduce flowers, such as roses, petunia, with altered colors.

Lignins are organic compounds essential for maintaining mechanicalstrength of cell walls in plants. Although essential, lignins have somedisadvantages. They cause indigestibility of sillage crops and areundesirable to paper production from wood pulp and others. Transgenicplants expressing ribozymes targeted against genes involved in ligninproduction such as, O-methyltransferase, cinnamoyl-CoA:NADPH reductaseor cinnamoyl alcohol dehydrogenase (Doorsselaere et al., 1995, The PlantJ. 8, 855; Atanassova et al., 1995, The Plant J. 8, 465; Shewmaker etal., supra; Dwivedi et al., 1994, Pl. Mol. Biol., 26, 61), would havealtered levels of lignin.

Other useful targets for useful ribozymes are disclosed in Draper etal., International PCT Publication No. WO 93/23569, Sullivan et al.,International PCT Publication No. WO 94/02595, as well as by Stinchcombet al., International PCT Publication No. WO 95/31541, and herebyincorporated by reference herein in totality.

Modulation of Granule Bound Starch Synthase Gene Expression in Plants

In plants, starch biosynthesis occurs in both chloroplasts (short termstarch storage) and in the amyloplast (long term starch storage). Starchgranules normally consist of a linear chain of α(1-4)-linked α-D-glucoseunits (amylose) and a branched form of amylose cross-linked by α(1-6)bonds (amylopectin). An enzyme involved in the synthesis of starch inplants is starch synthase which produces linear chains of α(1-4)-glucoseusing ADP-glucose. Two main forms of starch synthase are found inplants: granule bound starch synthase (GBSS) and a soluble form locatedin the stroma of chloroplasts and in amyloplasts (soluble starchsynthase). Both forms of this enzyme utilize ADP-D-glucose while thegranular bound form also utilizes UDP-D-glucose, with a preference forthe former. The GBSS, known as waxy protein, has a molecular mass ofbetween 55 to about 70 kDa in a variety of plants in which it has beencharacterized. Mutations affecting the GBSS gene in several plantspecies has resulted in the loss of amylose, while the total amount ofstarch has remained relatively unchanged. In addition to a loss of GBSSactivity, these mutants also contain altered, reduced levels, or no GBSSprotein (Mac Donald and Preiss, Plant Physiol. 78: 849-852 (1985), Sano,Theor. Appl. Genet. 68: 467-473 (1984), Hovenkamp-Hermelink et al.Theor. Appl. Genet. 75: 217-221 91987), Shure et al. Cell 35, 225-233(1983), Echt and Schwartz Genetics 99: 275-284 (1981)). The presence ofa branching enzyme as well as a soluble ADP-glucose starch glycosyltransferase in both GBSS mutants and wild type plants indicates theexistence of independent pathways for the formation of the branchedchain polymer amylopectin and the straight chain polymer amylose.

The Wx (waxy) locus encodes a granule bound glucosyl transferaseinvolved in starch biosynthesis. Expression of this enzyme is limited toendosperm, pollen and the embryo sac in maize. Mutations in this locushave been termed waxy due to the appearance of mutant kernels, which isthe phenotype resulting from an reduction in amylose composition in thekernels. In maize, this enzyme is transported into the amyloplast of thedeveloping endosperm where it catalyses production of amylose. Cornkernels are about 70% starch, of which 27% is linear amylose and 73% isamylopectin. Waxy is a recessive mutation in the gene encoding granulebound starch synthase (GBSS). Plants homozygous for this recessivemutation produce kernels that contain 100% of their starch in the formof amylopectin.

Ribozymes, with their catalytic activity and increased site specificity(as described below), represent more potent and perhaps more specificinhibitory molecules than antisense oligonucleotides. Moreover, theseribozymes are able to inhibit GBSS activity and the catalytic activityof the ribozymes is required for their inhibitory effect. For those ofordinary skill in the art, it is clear from the examples that otherribozymes may be designed that cleave target mRNAs required for GBSSactivity in plant species other than maize.

Thus, in a preferred embodiment, the invention features ribozymes thatinhibit enzymes involved in amylose production, e.g., by reducing GBSSactivity. These endogenously expressed RNA molecules contain substratebinding domains that bind to accessible regions of the target mRNA. TheRNA molecules also contain domains that catalyze the cleavage of RNA.The RNA molecules are preferably ribozymes of the hammerhead or hairpinmotif. Upon binding, the ribozymes cleave the target mRNAs, preventingtranslation and protein accumulation. In the absence of the expressionof the target gene, amylose production is reduced or inhibited. Specificexamples arc provided below infra.

Preferred embodiments include the ribozymes having binding arms whichare complementary to the binding sequences in Tables IIIA, VA and VB.Examples of such ribozymes are shown in Tables IIIB-V. Those in the artwill recognize that while such examples are designed to one plant's(e.g., maize) mRNA, similar ribozymes can be made complementary to otherplant species' mRNA. By complementary is thus meant that the bindingarms enable ribozymes to interact with the target RNA in asequence-specific manner to cause cleavage of a plant mRNA target.Examples of such ribozymes consist essentially of sequences shown inTables IIIB-V.

Preferred embodiments are the ribozymes and methods for their use in theinhibition of starch granule bound ADP (UDP)-glucose: α-1,4-D-glucan4-α-glucosyl transferase i.e., granule bound starch synthase (GBSS)activity in plants. This is accomplished through the inhibition ofgenetic expression, with ribozymes, which results in the reduction orelimination of GBSS activity in plants.

In another aspect of the invention, ribozymes that cleave targetmolecules and inhibit amylose production are expressed fromtranscription units inserted into the plant genome. Preferably, therecombinant vectors capable of stable integration into the plant genomeand selection of transformed plant lines expressing the ribozymes areexpressed either by constitutive or inducible promoters in the plantcells. Once expressed, the ribozymes cleave their target mRNAs andreduce amylose production of their host cells. The ribozymes expressedin plant cells are under the control of a constitutive promoter, atissue-specific promoter or an inducible promoter.

Modification of corn starch is an important application of ribozymetechnology which is capable of reducing specific gene expression. A highlevel of amylopectin is desirable for the wet milling process of cornand there is also some evidence that high amylopectin corn leads toincreased digestibility and therefore energy availability in feed.Nearly 10% of wet-milled corn has the waxy phenotype, but because of itsrecessive nature the traditional waxy varieties are very difficult forthe grower to handle Ribozymes targeted to cleave the GBSS mRNA and thusreduce GBSS activity in plants and in particular, corn endosperm willact as a dominant trait and produce corn plants with the waxy phenotypethat will be easier for the grower to handle.

Modification of Fatty Acid Saturation Profile in Plants

Fatty acid biosynthesis in plant tissues is initiated in thechloroplast. Fatty acids are synthesized as thioesters of acyl carrierprotein (ACP) by the fatty acid synthase complex (FAS). Fatty acids withchain lengths of 16 carbons follow one of three paths: they arereleased, immediately after synthesis, and transferred to glycerol3-phosphate (G3P) by a chloroplast acyl transferase for furthermodification within the chloroplast; 2) they are released andtransferred to Co-enzyme A (CoA) upon export from the plastid bythioesterases; or 3) they are further elongated to C18 chain lengths.The C18 chains are rapidly desaturated at the C9 position bystearoyl-ACP desaturase. This is followed by immediate transfer of theoleic acid (18:1) group to G3P within the chloroplast, or by export fromthe chloroplast and conversion to oleoyl-CoA by thioesterases(Somerville and Browse, 1991 Science 252: 80-87). The majority of C16fatty acids follow the third pathway.

In corn seed oil the predominant triglycerides are produced in theendoplasmic reticulum. Most oleic acids (18:1) and some palmitic acids(16:0) are transferred to G3P from phosphatidic acids, which are thenconverted to diacyl glycerides and phosphatidyl choline. Furtherdesaturation of the acyl chains on phosphatidyl choline by membranebound desaturases takes place in the endoplasmic reticulum. Di- andtri-unsaturated chains are then released into the acyl-CoA pool andtransferred to the C3 position of the glycerol backbone in diacylglycerol in the production of triglycerides (Frentzen, 1993 in LipidMetabolism in Plants., p.195-230, (ed. Moore, T. S.) CRC Press, BocaRaton, Fla.). A schematic of the plant fatty acid biosynthesis pathwayis shown in FIGS. 11 and 12. The three predominant fatty acids in cornseed oil are linoleic acid (18:2, ˜59%), oleic acid (18:1, ˜26%), andpalmitic acid (16:0, ˜11%). These are average values and may be somewhatdifferent depending on the genotype; however, composite samples of USCorn Belt produced oil analyzed over the past ten years haveconsistently had this composition (Glover and Mertz, 1987 in:Nutritional Quality of Cereal Grains: genetic and agronomicimprovement., p.183-336, (eds. Olson, R. A. and Frey, K. J.) Am. Soc.Agronomy. Inc. Madison, Wis.; Fitch-Haumann, 1985 J. Am. Oil Chem. Soc.62: 1524-1531; Strecker et al., 1990 in Edible fats and oils processing:basic principles and modern practices (ed. Erickson, D. R.) Am. OilChemists Soc. Champaign, Ill.). This predominance of C18 chain lengthsmay reflect the abundance and activity of several key enzymes, such asthe fatty acid synthase responsible for production of C18 carbon chains,the stearoyl-ACP desaturase (Δ-9 desaturase) for production of 18:1 anda microsomal Δ-12 desaturase for conversion of 18:1 to 18:2.

Δ-9 desaturase (also called stearoyl-ACP desaturase) of plants is asoluble chloroplast enzyme which uses C18 and occasionally C16-acylchains linked to acyl carrier protein (ACP) as a substrate (McKeon, T.A. and Stumpf, P. K., 1982 J. Biol. Chem. 257: 12141-12147). Thiscontrasts to the mammalian, lower eukaryotic and cyanobacterial Δ-9desaturases. Rat and yeast Δ-9 desaturases are membrane bound microsomalenzymes using acyl-CoA chains as substrates, whereas cyanobacterial Δ-9desaturase uses acyl chains on diacyl glycerol as substrate. To dateseveral Δ-9 desaturase cDNA clones from dicotelydenous plants have beenisolated and characterized (Shanklin and Somerville, 1991 Proc. Natl.Acad. Sci. USA 88: 2510-2514; Knutzon et al., 1991 Plant Physiol. 96:344-345; Sato et al., 1992 Plant Physiol. 99: 362-363; Shanklin et al.,1991 Plant Physiol. 97: 467-468; Slocombe et al., 1992 Plant. Mol. Biol.20: 151-155; Tayloret al., 1992 Plant Physiol. 100: 533-534; Thompson etal., 1991 Proc. Natl. Acad. Sci. USA 88: 2578-2582). Comparison of thedifferent plant Δ-9 desaturase sequences suggests that this is a highlyconserved enzyme, with high levels of identity both at the amino acidlevel (˜90%) and at the nucleotide level (˜80%). However, as might beexpected from its very different physical and enzymological properties,no sequence similarity exists between plant and other Δ-9 desaturases(Shanklin and Somerville, supra).

Purification and characterization of the castor bean desaturase (andothers) indicates that the Δ-9 desaturase is active as a homodimer; thesubunit molecular weight is ˜41 kDa. The enzyme requires molecularoxygen, NADPH, NADPH ferredoxin oxidoreductase and ferredoxin foractivity in vitro. Fox et al. , 1993 (Proc. Natl. Acad. Sci. USA 90:2486-2490) showed that upon expression in E. coli the castor bean enzymecontains four catalytically active ferrous atoms per homodimer. Theoxidized enzyme contains two identical diferric clusters, which in thepresence of dithionite are reduced to the diferrous state. In thepresence of stearoyl-CoA and O₂ the clusters return to the diferricstate. This suggests that the desaturase belongs to a group of O₂activating proteins containing diiron-oxo clusters. Other members ofthis group are ribonucleotide reductase and methane monooxygenasehydroxylase. Comparison of the predicted primary structure for thesecatalytically diverse proteins shows that all contain a conserved pairof amino acid sequences (Asp/Glu)-Glu-Xaa-Arg-His separated by ˜80-100amino acids.

Traditional plant breeding programs have shown that increased stearatelevels can be achieved without deleterious consequences to the plant. Insafflower (Ladd and Knowles, 1970 Crop Sci. 10: 525-527) and in soybean(Hammond and Fehr, 1984 J. Amer. Oil Chem. Soc. 61: 1713-1716; Graef etal., 1985 Crop Sci. 25: 1076-1079) stearate levels have been increasedsignificantly. This demonstrates the flexibility in fatty acidcomposition of seed oil.

Increases in Δ-9 desaturase activity have been achieved by thetransformation of tobacco with the Δ-9 desaturase genes from yeast(Polashock et al., 1992 Plant Physiol. 100, 894) or rat (Grayburn et.al., 1992 BioTechnology 10, 675). Both sets of transgenic plants hadsignificant changes in fatty acid composition, yet were phenotypicallyidentical to control plants.

Corn (maize) has been used minimally for the production of margarineproducts because it has traditionally not been utilized as an oil cropand because of the relatively low seed oil content when compared withsoybean and canola. However, corn oil has low levels of linolenic acid(18:3) and relatively high levels of palmitic (16:0) acid (desirable inmargarine). Applicant believes that reduction in oleic and linoleic acidlevels by down-regulation of Δ-9 desaturase activity will make corn aviable alternative to soybean and canola in the saturated oil market.

Margarine and confectionary fats are produced by chemical hydrogenationof oil from plants such as soybean. This process adds cost to theproduction of the margarine and also causes both cis and trans isomersof the fatty acids. Trans isomers are not naturally found in plantderived oils and have raised a concern for potential health risks.Applicant believes that one way to eliminate the need for chemicalhydrogenation is to genetically engineer the plants so that desaturationenzymes are down-regulated. Δ-9 desaturase introduces the first doublebond into 18 carbon fatty acids and is the key step effecting the extentof desaturation of fatty acids.

Thus, in a preferred embodiment, the invention concerns compositions(and methods for their use) for the modification of fatty acidcomposition in plants. This is accomplished through the inhibition ofgenetic expression, with ribozymes, antisense nucleic acid,cosuppression or triplex DNA, which results in the reduction orelimination of certain enzyme activities in plants, such as Δ-9desaturase. Such activity is reduced in monocotyledon plants, such asmaize, wheat, rice, palm, coconut and others. Δ-9 desaturase activitymay also be reduced in dicotyledon plants such as sunflower, safflower,cotton, peanut, olive, sesame, cuphea, flax, jojoba, grape and others.

Thus, in one aspect, the invention features ribozymes that inhibitenzymes involved in fatty acid unsaturation, e.g., by reducing Δ-9desaturase activity. These endogenously expressed RNA molecules containsubstrate binding domains that bind to accessible regions of the targetmRNA. The RNA molecules also contain domains that catalyze the cleavageof RNA. The RNA molecules are preferably ribozymes of the hammerhead orhairpin motif. Upon binding, the ribozymes cleave the target mRNAs,preventing translation and protein accumulation. In the absence of theexpression of the target gene, stearate levels are increased andunsaturated fatty acid production is reduced or inhibited. Specificexamples are provided below in the Tables listed directly below.

In preferred embodiments, the ribozymes have binding arms which arecomplementary to the sequences in the Tables VI and VIII. Those in theart will recognize that while such examples are designed to one plant's(e.g., corn) mRNA, similar ribozymes can be made complementary to otherplant's mRNA. By complementary is thus meant that the binding arms ofthe ribozymes are able to interact with the target RNA in asequence-specific manner and enable the ribozyme to cause cleavage of aplant mRNA target. Examples of such ribozymes are typically sequencesdefined in Tables VII and VIII. The active ribozyme typically containsan enzymatic center equivalent to those in the examples, and bindingarms able to bind plant mRNA such that cleavage at the target siteoccurs. Other sequences may be present which do not interfere with suchbinding and/or cleavage.

The sequences of the ribozymes that are particularly useful in thisstudy, are shown in Tables VII and VIII.

Those in the art will recognize that ribozyme sequences listed in theTables are representative only of many more such sequences where theenzymatic portion of the ribozyrne (all but the binding arms) is alteredto affect activity. For example, stem-loop II sequence of hammerheadribozymes listed in Table IV (5′-GGCGAAAGCC-3′)(SEQ ID NO. 1237) can bealtered (substitution, deletion, and/or insertion) to contain anysequences, preferably provided that a minimum of a two base-paired stemstructure can form. Similarly, stem-loop IV sequence of hairpinribozymes listed in Tables V and VIII (5′-CACGUUGUG-3′) (SEQ ID NO.1238) can be altered (substitution, deletion, and/or insertion) tocontain any sequence, preferably provided that a minimum of a twobase-paired stem structure can form. Such ribozymes are equivalent tothe ribozymes described specifically in the Tables.

In another aspect of the invention, ribozymes that cleave targetmolecules and reduce unsaturated fatty acid content in plants areexpressed from transcription units inserted into the plant genome.Preferably, the recombinant vectors capable of stable integration intothe plant genome and selection of transformed plant lines expressing theribozymes are expressed either by constitutive or inducible promoters inthe plant cells. Once expressed, the ribozymes cleave their target mRNAsand reduce unsaturated fatty acid production of their host cells. Theribozymes expressed in plant cells are under the control of aconstitutive promoter, a tissue-specific promoter or an induciblepromoter.

Modification of fatty acid profile is an important application ofnucleic acid-based technologies which are capable of reducing specificgene expression. A high level of saturated fatty acid is desirable inplants that produce oils of commercial importance.

In a related aspect, this invention features the isolation of the cDNAsequence encoding Δ-9 desaturase in maize.

In preferred embodiments, hairpin and hammerhead ribozymes that cleaveΔ-9 desaturase mRNA are also described. Those of ordinary skill in theart will understand from the examples described below that otherribozymes that cleave target mRNAs required for Δ-9 desaturase activitymay now be readily designed and are within the scope of the invention.

While specific examples to corn RNA are provided, those in the art willrecognize that the teachings are not limited to corn. Furthermore, thesame target may be used in other plant species. The complementary armssuitable for targeting the specific plant RNA sequences are utilized inthe ribozyme targeted to that specific RNA. The examples and teachingsherein are meant to be non-limiting, and those skilled in the art willrecognize that similar embodiments can be readily generated in a varietyof different plants to modulate expression of a variety of differentgenes, using the teachings herein, and are within the scope of theinventions.

Standard molecular biology techniques were followed in the examplesherein. Additional information may be found in Sambrook, J., Fritsch, E.F., and Maniatis, T. (1989), Molecular Cloning a Laboratory Manual,second edition, Cold Spring Harbor: Cold Spring Harbor Laboratory Press,which is incorporated herein by reference.

EXAMPLES Example 1 Isolation of Δ9 Desaturase cDNA from Zea mays

Degenerate PCR primers were designed and synthesized to two conservedpeptides involved in diiron-oxo group binding of plant Δ-9 desaturases.A 276 bp DNA fragment was PCR amplified from maize embryo cDNA and wascloned in to a vector. The predicted amino acid sequence of thisfragment was similar to the sequence of the region separated by the twoconserved peptides of dicot Δ-9 desaturase proteins. This was used toscreen a maize embryo cDNA library. A total of 16 clones were isolated;further restriction mapping and hybridization identified one clone whichwas sequenced. Features of the cDNA insert are: a 1621 nt cDNA; 145 nt5′ and 294 nt 3′ untranslated regions including a 18 nt poly A tail; a394 amino acid open reading frame encoding a 44.7 kD polypeptide; and85% amino acid identity with castor bean Δ-9 desaturase gene for thepredicted mature protein. The complete sequence is presented in FIG. 10.

Example 2 Identification of Potential Ribozyme Cleavage Sites for Δ9Desaturase

Approximately two hundred and fifty HH ribozyme sites and approximatelyforty three HP sites were identified in the corn Δ-9 desaturase mRNA. AHH site consists of a uridine and any nucleotide except guanosine (UH).Tables VI and VIII have a list of HH and HP ribozyme cleavage sites. Thenumbering system starts with 1 at the 5′ end of a Δ-9 desaturase cDNAclone having the sequence shown in FIG. 10.

Ribozymes, such as those listed in Tables VII and VIII, can be readilydesigned and synthesized to such cleavage sites with between 5 and 100or more bases as substrate binding arms (see FIGS. 1-5). These substratebinding arms within a ribozyme allow the ribozyme to interact with theirtarget in a sequence-specific manner.

Example 3 Selection of Ribozyme Cleavage Sites for Δ9 Desaturase

The secondary structure of Δ-9 desaturase mRNA was assessed by computeranalysis using algorithms, such as those developed by M. Zuker (Zuker,M., 1989 Science, 244, 48-52). Regions of the mRNA that did not formsecondary folding structures with RNA/RNA stems of over eightnucicotides and contained potential hammerhead ribozyme cleavage siteswere identified.

These sites were assessed for oligonucleotide accessibility by RNase Hassays (see Example 4 infra).

Example 4 RNaseH Assays for Δ9 Desaturase

Forty nine DNA oligonucleotides, each twenty one nucleotides long wereused in RNase H assays. These oligonucleotides covered 108 sites withinΔ-9 desaturase RNA. RNase H assays (FIG. 6) were performed using a fulllength transcript of the Δ-9 desaturase cDNA. RNA was screened foraccessible cleavage sites by the method described generally in Draper etal., supra. Briefly, DNA oligonucleotides representing ribozyme cleavagesites were synthesized. A polymerase chain reaction was used to generatea substrate for T7 RNA polymerase transcription from corn cDNA clones.Labeled RNA transcripts were synthesized in vitro from these templates.The oligonucleotides and the labeled transcripts were annealed, RNAseHwas added and the mixtures were incubated for 10 minutes at 37° C.Reactions were stopped and RNA separated on sequencing polyacrylamidegels. The percentage of the substrate cleaved was determined byautoradiographic quantitation using a Molecular Dynamics phosphorimaging system (FIGS. 13 and 14).

Example 5 Hammerhead and Hairpin Ribozymes for Δ9 Desaturase

Hammerhead (HH) and hairpin (HP) ribozymes were designed to the sitescovered by the oligos which cleaved best in the RNase H assays. Theseribozymes were then subjected to analysis by computer folding and theribozymes that had significant secondary structure were rejected.

The ribozymes were chemically synthesized. The general procedures forRNA synthesis have been described previously (Usman et al., 1987, J. Am.Chem. Soc., 109, 7845-7854 and in Scaringe et al., 1990, Nucl. AcidsRes., 18, 5433-5341; Wincott et al., 1995, Nucleic Acids Res. 23, 2677).Small scale syntheses were conducted on a 394 Applied Biosystems, Inc.synthesizer using a modified 2.5 μmol scale protocol with a 5 mincoupling step for alkylsilyl protected nucleotides and 2.5 min couplingstep for 2′-O-methylated nucleotides. Table II outlines the amounts, andthe contact times, of the reagents used in the synthesis cycle. A6.5-fold excess (163 μL of 0.1 M=16.3 μmol) of phosphoramidite and a24-fold excess of S-ethyl tetrazole (238 μL of 0.25 M=59.5 μmol)relative to polymer-bound 5′-hydroxyl was used in each coupling cycle.Average coupling yields on the 394, determined by colorimetricquantitation of the trityl fractions, was 97.5-99%. Otheroligonucleotide synthesis reagents for the 394: Detritylation solutionwas 2% TCA in methylene chloride (ABI); capping was performed with 16%N-Methyl imidazole in THF (ABI) and 10% acetic anhydride/10%2,6-lutidine in THF (ABI); oxidation solution was 16.9 mM 12, 49 mMpyridine, 9% water in THF (Millipore). B & J Synthesis Gradeacetonitrile was used directly from the reagent bottle. S-Ethyltetrazole solution (0.25 M in acetonitrile) was made up from the solidobtained from American International Chemical, Inc.

Deprotection of the RNA was performed as follows. The polymer-boundoligoribonucleotide, trityl-off, was transferred from the synthesiscolumn to a 4 mL glass screw top vial and suspended in a solution ofmethylamine (MA) at 65° C. for 10 min. After cooling to −20° C., thesupernatant was removed from the polymer support. The support was washedthree times with 1.0 mL of EtOH:MeCN:H₂O/3:1:1, vortexed and thesupernatant was then added to the first supernatant. The combinedsupernatants, containing the oligoribonucleotide, were dried to a whitepowder.

The base-deprotected oligoribonucleotide was resuspended in anhydrousTEA•HF/NMP solution (250 μL of a solution of 1.5 mLN-methylpyrrolidinone, 750 μL TEA and 1.0 mL TEA•3HF to provide a 1.4 MHF concentration) and heated to 65° C. for 1.5 h. The resulting, fullydeprotected, oligomer was quenched with 50 mM TEAB (9 mL) prior to anionexchange desalting.

For anion exchange desalting of the deprotected oligomer, the TEABsolution was loaded onto a Qiagen 500® anion exchange cartridge (QiagenInc.) that was prewashed with 50 mM TEAB (10 mL). After washing theloaded cartridge with 50 mM TEAB (10 mL), the RNA was eluted with 2 MTEAB (10 mL) and dried down to a white powder.

Inactive hammerhead ribozymes were synthesized by substituting a U forG₅ and a U for A₁₄ (numbering from (Hertel, K. J., et al., 1992, NucleicAcids Res., 20, 3252).

The hairpin ribozymes were synthesized as described above for thehammerhead RNAs.

Ribozymes were also synthesized from DNA templates using bacteriophageT7 RNA polymerase (Milligan and Uhlenbeck, 1989, Methods Enzymol. 180,51). Ribozymes were purified by gel electrophoresis using generalmethods or were purified by high pressure liquid chromatography (HPLC;See Wincott et al., 1996, supra, the totality of which is herebyincorporated herein by reference) and were resuspended in water. Thesequences of the chemically synthesized ribozymes used in this study areshown below in Tables VII and VIII.

Example 6 Long Substrate Tests for Δ9 Desaturase Ribozymes

Target RNA used in this study was 1621 nt long and contained cleavagesites for all the HH and HP ribozymes targeted against Δ-9 desaturaseRNA. A template containing T7 RNA polymerase promoter upstream of Δ-9desaturase target sequence, was PCR amplified from a cDNA clone. TargetRNA was transcribed from this PCR amplified template using T7 RNApolymerase. The transcript was internally labeled during transcriptionby including [α-³²P] CTP as one of the four ribonucleotidetriphosphates. The transcription mixture was treated with DNase-I,following transcription at 37° C. for 2 hours, to digest away the DNAtemplate used in the transcription. The transcription mixture wasresolved on a denaturing polyacrylamide gel. Bands corresponding tofull-length RNA was isolated from a gel slice and the RNA wasprecipitated with isopropanol and the pellet was stored at 4° C.

Ribozyme cleavage reactions were carried out under ribozyme excess(k_(cat)/K_(M)) conditions (Herschlag and Cech, 1990, Biochemistry 29,10159-10171). Briefly, 1 mM ribozyme and <10 nM internally labeledtarget RNA were denatured separately by heating to 65° C. for 2 min inthe presence of 50 mM Tris.HCl, pH 7.5 and 10 mM MgCl₂. The RNAs wererenatured by cooling to the reaction temperature (37° C., 26° C. or 20°C.) for 10-20 min. Cleavage reaction was initiated by mixing theribozyme and target RNA at appropriate reaction temperatures. Aliquotswere taken at regular intervals of time and the reaction was quenched byadding equal volume of stop buffer. The samples were resolved on 4%sequencing gel.

The results from ribozyme cleavage reactions, at 26° C. or 20° C., aresummarized in Table IX and FIGS. 15 and 16. Of the ribozymes tested,seven hammerheads and two hairpins showed significant cleavage of Δ-9desaturase RNA (FIGS. 15 and 16). Ribozymes to other sites showed variedlevels of activity.

Example 7 Cleavage of the Target RNA Using Multiple RibozymeCombinations for Δ9 Desaturase

Several of the above ribozymes were incorporated into a multimerribozyme construct which contains two or more ribozymes embedded in acontiguous stretch of complementary RNA sequence. Non-limiting examplesof multimer ribozymes are shown in FIGS. 17, 18, 19 and 23. Theribozymes were made by annealling complementary oligonucleotides andcloning into an expression vector containing the Cauliflower MosaicVirus 35S enhanced promoter (Franck et al., 1985 Cell 21, 285), themaize Adh 1 intron (Dennis et al., 1984 Nucl. Acids Res. 12, 3983) andthe Nos polyadenylation signal (DePicker et al., 1982 J. Molec. Appl.Genet. 1, 561). Cleavage assays with T7 transcripts made from thesemultimer-containing transcription units are shown in FIGS. 20 and 21.These are non-limiting examples; those skilled in the art will recognizethat similar embodiments, consisting of other ribozyme combinations,introns and promoter elements, can be readily generated using techniquesknown in the art and are within the scope of this invention.

Example 8 Construction of Ribozyme Expressing Transcription Units for Δ9Desaturase

Ribozymes targeted to cleave Δ-9 desaturase mRNA are endogenouslyexpressed in plants, either from genes inserted into the plant genome(stable transformation) or from episomal transcription units (transientexpression) which are part of plasmid vectors or viral sequences. Theseribozymes can be expressed via RNA polymerase I, II, or III plant orplant virus promoters (such as CaMV). Promoters can be eitherconstitutive, tissue specific, or developmentally expressed.

Δ9 259 Monomer Ribozyme Constructs (RPA 114, 115)

These are the Δ-9 desaturase 259 monomer hammerhead ribozyme clones. Theribozymes were designed with 3 bp long stem II and 20 bp (total) longsubstrate binding arms targeted against site 259. The active version isRPA114, the inactive is RPA 115. The parent plasmid, pDAB367, wasdigested with Not I and filled in with Klenow to make a blunt acceptorsite. The vector was then digested with Hind III restriction enzyme. Theribozyme containing plasmids were cut with Eco RI, filled-in with Klenowand recut with Hind III. The insert containing the entire ribozymetranscription unit was gel-purified and ligated into the pDAB 367vector. The constructs are checked by digestion with SgfI/Hind III andXba I/Sst I and confirmed by sequencing.

Δ9 453 Multimer Ribozyme Constructs (RPA 118, 119)

These are the Δ-9 desaturase 453 Multimer hammerhead ribozyme clones(see FIG. 17). The ribozymes were designed with 3 bp long stem IIregions. Total length of the substrate binding anms of the multimerconstruct was 42 bp. The active version is RPA 118, the inactive is 119.The constructs were made as described above for the 259 monomer. Themultimer construct was designed with four hammerhead ribozymes targetedagainst sites 453, 464, 475 and 484 within Δ-9 desaturase RNA.

Δ9 252 Multimer Ribozyme Constructs (RPA 85, 113)

These are the Δ-9 desaturase 252 multimer ribozyme clones placed at the3′ end of bar (phosphoinothricin acetyl transferase; Thompson et al.,1987 EMBO J. 6: 2519-2523) open reading frame. The multimer contructswere designed with 3 bp long stem II regions. Total length of thesubstrate binding arms of the multimer construct was 91 bp. RPA 85 isthe active ribozyme, RPA 113 is the inactive. The vector was constructedas follows: The parent plasmid pDAB 367 was partially digested with BglII and the single cut plasmid was gel-purified. This was recut with EcoRI and again gel-purified to isolate the correct Bgl II/Eco RI cutfragment. The Bam HI/Eco RI inserts from the ribozyme constructs weregel-isolated (this contains the ribozyme and the NOS poly A region) andligated into the 367 vector. The identitiy of positive plasmids wereconfinned by performing a Nco I/Sst I digest and sequencing.

Useful transgenic plants can be identified by standard assays. Thetransgenic plants can be evaluated for reduction in Δ-9 desaturaseexpression and Δ-9 desaturase activity as discussed in the examplesinfra.

Example 9 Identification of Potential Ribozyme Cleavage Sites in GBSSRNA

Two hundred and forty one hammer-head ribozyme sites were identified inthe corn GBSS mRNA polypeptide coding region (see table IIIA). Ahammer-head site consists of a uridine and any nucleotide except guanine(UH). Following is the sequence of GBSS coding region for corn (SEQ.I.D. No. 25). The numbering system starts with 1 at the 5′ end of a GBSScDNA clone having the following sequence (5′ to 3′):

1 72 (SEQ I.D. NO. 25).GACCGATCGATCGCCACAGCCAACACCACCCGCCGAGGCGACGCGACAGCCGCCAGGAGGAAGGAATAAACT73 144CACTGCCAGCCAGTGAAGGGGGAGAAGTGTACTGCTCCGTCCACCAGTGCGCGCACCGCCCGGCAGGGCTGC145 216TCATCTCGTCGACGACCAGTGGATTAATCGGCATGGCGGCTCTAGCCACGTCGCAGCTCGTCGCAACGCGCG217 288CCGGCCTGGGCGTCCCGGACGCGTCCACGTTCCGCCGCGGCGCCGCGCAGGGCCTGAGGGGGGGCCGGACGG289 360CGTCGGCGGCGGACACGCTCAGCATTCGGACCAGCGCGCGCGCGGCGCCCAGGCTCCAGCACCAGCAGCAGC361 432AGCAGGCGCGCCGCGGGGCCAGGTTCCCGTCGCTCGTCGTGTGCGCCAGCGCCGGCATGAACGTCGTCTTCG433 504TCGGCGCCGAGATGGCGCCGTGGAGCAAGACCGGCGGCCTCGGCGACGTCCTCGGCGGCCTGCCGCCGGCCA505 576TGGCCGCGAATGGGCACCGTGTCATGGTCGTCTCTCCCCGCTACGACCAGTACAAGGACGCCTGGGACACCA577 648GCGTCGTGTCCGAGATCAAGATGGGAGACAGGTACGAGACGGTCAGGTTCTTCCACTGCTACAAGCGCGGAG649 720TGGACCGCGTGTTCGTTGACCACCCACTGTTCCTGGAGAGGGTTTGGGGAAAGACCGAGGAGAAGATCTACG721 792GGCCTGACGCTGGAACGGACTACAGGGACAACCAGCTGCGGTTCAGCCTGCTATGCCAGGCAGCACTTGAAG793 864CTCCAAGGATCCTGAGCCTCAACAACAACCCATACTTCTCCGGACCATACGGGGAGGACGTCGTGTTCGTCT865 936GCAACGACTGGCACACCGGCCCTCTCTCGTGCTACCTCAAGAGCAACTACCAGTCCCACGGCATCTACAGGG937 1008ACGCAAAGACCGCTTTCTGCATCCACAACATCTCCTACCAGGGCCGGTTCGCCTTCTCCGACTACCCGGAGC1009 1080TGAACCTCCCGGAGAGATTCAAGTCGTCCTTCGATTTCATCGACGGCTACGAGAAGCCCGTGGAAGGCCGGA1081 1152AGATCAACTGGATGAAGGCCGGGATCCTCGAGGCCGACAGGGTCCTCACCGTCAGCCCCTACTACGCCGAGG1153 1224AGCTCATCTCCGGCATCGCCAGGGGCTGCGAGCTCGACAACATCATGCGCCTCACCGGCATCACCGGCATCG1225 1296TCAACGGCATGGACGTCAGCGAGTGGGACCCCAGCAGGGACAAGTACATCGCCGTGAAGTACGACGTGTCGA1297 1368CGGCCGTGGAGGCCAAGGCGCTGAACAAGGAGGCGCTGCAGGCGGAGGTCGGGCTCCCGGTGGACCGGAACA1369 1440TCCCGCTGGTGGCGTTCATCGGCAGGCTGGAAGAGCAGAAGGGACCCGACGTCATGGCGGCCGCCATCCCGC1441 1512AGCTCATGGAGATGGTGGAGGACGTGCAGATCGTTCTGCTGGGCACGGGCAAGAAGAAGTTCGAGCGCATGC1513 1584TCATGAGCGCCGAGGAGAAGTTCCCAGGCAAGGTGCGCGCCGTGGTCAAGTTCAACGCGGCGCTGGCGCACC1585 1656ACATCATGGCCGGCGCCGACGTGCTCGCCGTCACCAGCCGCTTCGAGCCCTGCGGCCTCATCCAGCTGCAGG1657 1728GGATGCGATACGGAACGCCCTGCGCCTGCGCGTCCACCGGTGGACTCGTCGACACCATCATCGAAGGCAAGA1729 1800CCGGGTTCCACATGGGCCGCCTCAGCGTCGACTGCAACGTCGTGGAGCCGGCGGACGTCAAGAAGGTGGCCA1801 1872CCACCTTGCAGCGCGCCATCAAGGTGGTCGGCACGCCGGCGTACGAGGAGATGGTGAGGAACTGCATGATCC1873 1944AGGATCTCTCCTGGAAGGGCCCTGCCAAGAACTGGGAGAACGTGCTGCTCAGCCTCGGGGTCGCCGGCGGCG1945 2016AGCCAGGGGTCGAAGGCGAGGAGATCGCGCCGCTCGCCAAGGAGAACGTGGCCGCGCCCTGAAGAGTTCGGC2017 2088CTGCAGGCCCCCTGATCTCGCGCGTGGTGCAAACATGTTGGGACATCTTCTTATATATGCTGTTTCGTTTAT2089 2160GTGATATGGACAAGTATGTGTAGCTGCTTGCTTGTGCTAGTGTAATATAGTGTAGTGGTGGCCAGTGGCACA2161 2232ACCTAATAAGCGCATGAACTAATTGCTTGCGTGTGTAGTTAAGTACCGATCGGTAATTTTATATTGCGAGTA2233 AATAAATGGACCTGTAGTGGTGGAAAAAAAAAAAA

There are approximately 53 potential hairpin ribozyme sites in the GBSSmRNA. The ribozyme and target sequences are listed in Table V.

Ribozymes can be readily designed and synthesized to such sites withbetween 5 and 100 or more bases as substrate binding arms (see FIGS.1-5) as described above.

Example 10 Selection of Ribozyme Cleavage Sites for GBSS

The secondary structure of GBSS mRNA was assessed by computer analysisusing folding algorithms, such as the ones developed by M. Zuker (Zuker,M., 1989 Science, 244, 48-52. Regions of the mRNA that did not formsecondary folding structures with RNA/RNA stems of over eightnucleotides and contained potential hammerhead ribozyme cleavage siteswere identified.

These sites which were then assessed for oligonucleotide accessibilitywith RNase H assays (see FIG. 6). Fifty-eight DNA oligonucleotides, eachtwenty one nucleotides long were used in these assays. Theseoligonucleotides covered 85 sites. The position and designation of theseoligonucleotides were 195, 205, 240, 307, 390, 424, 472, 481, 539, 592,625, 636, 678, 725, 741, 811, 859, 891, 897, 912, 918, 928, 951, 958,969, 993, 999, 1015, 1027, 1032, 1056, 1084, 1105, 1156, 1168, 1186,1195, 1204, 1213, 1222, 1240, 1269, 1284, 1293, 1345, 1351, 1420, 1471,1533, 1563, 1714, 1750, 1786, 1806, 1819, 1921, 1954, and 1978.Secondary sites were also covered and included 202, 394, 384, 385, 484,624, 627, 628, 679, 862, 901, 930, 950, 952, 967, 990, 991, 1026, 1035,1108, 1159, 1225, 1273, 1534, 1564, 1558, and 1717.

Example 11 RNaseH Assays for GBSS

RNase H assays (FIG. 7) were performed using a full length transcript ofthe GBSS coding region, 3′ noncoding region, and part of the 5′noncoding region. The GBSS RNA was screened for accessible cleavagesites by the method described generally in Draper et al., supra. herebyincorporated by reference herein. Briefly, DNA oligonucleotidesrepresenting hammerhead ribozyme cleavage sites were synthesized. Apolymerase chain reaction was used to generate a substrate for T7 RNApolymerase transcription from corn cDNA clones. Labeled RNA transcriptswere synthesized in vitro from these templates. The oligonucleotides andthe labeled transcripts were annealed, RNAseH was added and the mixtureswere incubated for 10 minutes at 37° C. Reactions were stopped and RNAseparated on sequencing polyacrylamide gels. The percentage of thesubstrate cleaved was determined by autoradiographic quantitation usinga phosphor imaging system (FIG. 7).

Example 12 Hammerhead Ribozymes for GBSS

Hammerhead ribozymes with 10/10 (i.e., able to form 10 base pairs oneach arm of the ribozyme) nucleotide binding arms were designed to thesites covered by the oligos which cleaved best in the RNase H assays.These ribozymes were then subjected to analysis by computer folding andthe ribozymes that had significant secondary structure were rejected. Asa result of this screening procedure 23 ribozymes were designed to themost open regions in the GBSS mRNA, the sequences of these ribozymes areshown in Table IV.

The ribozymes were chemically synthesized. The method of synthesis usedfollows the procedure for normal RNA synthesis as described above (andin Usman et al., supra, Scaringe et al., and Wincott et al., supra) andare incorporated by reference herein, and makes use of common nucleicacid protecting and coupling groups, such as dimethoxytrityl at the5′-end, and phosphoramidites at the 3′-end. The average stepwisecoupling yields were >98%. Inactive ribozymes were synthesized bysubstituting a U for G₅ and a U for A₁₄ (numbering from (Hertel et al.,supra). Hairpin ribozymes were synthesized in two parts and annealed toreconstruct the active ribozyme (Chowrira and Burke, 1992, Nucleic AcidsRes., 20, 2835-). All ribozymes were modified to enhance stability bymodification of five ribonucleotides at both the 5′ and 3′ ends with2′-O-methyl groups. Ribozymes were purified by gel electrophoresis usinggeneral methods. (Ausubel et al., 1990 Current Protocols in MolecularBiology Wiley & Sons, NY) or were purified by high pressure liquidchromatography, as described above and were resuspended in water.

Example 13 Long Substrate Tests for GBSS

Target RNA used in this study was 900 nt long and contained cleavagesites for all the 23 HH ribozymes targeted against GBSS RNA. A templatecontaining T7 RNA polymerase promoter upstream of GBSS target sequence,was PCR amplified from a cDNA clone. Target RNA was transcribed fromthis PCR amplified template using T7 RNA polymerase. The transcript wasinternally labeled during transcription by including [α-³²P] CTP as oneof the four ribonucleotide tripbospbates. The transcription mixture wastreated with DNase-1, following transcription at 37° C. for 2 hours, todigest away the DNA template used in the transcription. Thetranscription mixture was resolved on a denaturing polyacrylamide gel.Bands corresponding to full-length RNA was isolated from a gel slice andthe RNA was precipitated with isopropanol and the pellet was stored at4° C.

Ribozyme cleavage reactions were carried out under ribozyme excess(k_(cat)/K_(M)) conditions (Herschlag and Cech, supra). Briefly, 1000 nMribozyme and <10 nM internally labeled target RNA were denaturedseparately by heating to 90° C. for 2 min. in the presence of 50 mMTris.HCl, pH 7.5 and 10 mM MgCl₂. The RNAs were renatured by cooling tothe reaction temperature (37° C., 26° C. and 20° C.) for 10-20 min.Cleavage reaction was initiated by mixing the ribozyme and target RNA atappropriate reaction temperatures. Alquots were taken at regularintervals of time and the reaction was quenched by adding equal volumeof stop buffer. The samples were resolved on 4% sequencing gel.

The results from ribozyme cleavage reactions, at the three differenttemperatures, summarized in FIG. 8. Seven lead ribozymes were chosen(425, 892, 919, 959, 968, 1241, and 1787). One of the active ribozymes(811) produced a strange pattern of cleavage products; as a result, itwas not chosen as one of our lead ribozymes.

Example 14 Cleavage of the GBSS RNA Using Multiple Ribozyme Combinations

Four of the lead ribozymes (892, 919, 959, 1241) were incubated withinternally labeled target RNA in the following combinations: 892 alone;892+919; 892+919+959; 892+919+959+1241. The fraction of RNA cleavageincreased in an additive manner with an increase in the number ofribozymes used in the cleavage reaction (FIG. 9). Ribozyme cleavagereactions were carried out at 20° C. as described above. These datasuggest that multiple ribozymes targeted to different sites on the samemRNA will increase the reduction of target RNA in an additive manner.

Example 15 Construction of Ribozvme Expressing Transcription Units forGBSS

Cloning of GBSS Multimer Ribozymes RPA 63 (active) and RPA 64 (inactive)A multimer ribozyme was constructed which contained four hammerheadribozymes targeting sites 892, 919, 959 and 968 of the GBSS mRNA. TwoDNA oligonucleotides (Macromolecular Resourses, Fort Collins, Colo.)were ordered which overlap by 18 nucleotides. The sequences were asfollows:

Oligo 1: CGC GGA TCC TGG TAG GAC TGA TGA GGC CGA AAG GCC GAA ATG TTG TGCTGA TGA GGC CGA AAG GCC GAA ATG CAG AAA GCG GTC TTT GCG TCC CTG TAG ATGCCG TGG C (SEQ ID NO. 1238)

Oligo 2: CGC GAG CTC GGC CCT CTC TTT CGG CCT TTC GGC CTC ATC AGG TGC TACCTC AAG AGC AAC TAC CAG TTT CGG CCT TTC GGC CTC ATC AGC CAC GGC ATC TACAGG G (SEQ ID NO. 1239)

Inactive versions of the above were made by substituting T for G5 and Tfor A14 within the catalytic core of each ribozyme motif.

These were annealed in 1 × Klenow Buffer (Gibco/BRL) at 90° C. for 5minutes and slow cooled to room temperature (22° C.). NTPs were added to0.2 mM and the oligos extended with Klenow enzyme at 1 unit/ul for onehour at 37° C. This was phenol/chloroform extracted and ethanolprecipitated, then resuspended in 1×React 3 buffer (Gibco/BRL) anddigested with Bam HI and Sst I for 1 hour at 37° C. The DNA was gelpurified on a 2% agarose gel using the Qiagen gel extraction kit.

The DNA fragments were ligated into BamHI/Sst I digested pDAB 353. Theligation was transformed into competent DH5α F′ bacteria (Gibco/BRL).Potential clones were screened by digestion with Bam HI/Eco RI. Cloneswere confirmed by sequencing. The total length of homology with thetarget sequence is 96 nucleotides.

919 Monomer Ribozyme (RPA 66)

A single ribozyme to site 919 of the GBSS mRNA was constructed with10/10 arms as follows. Two DNA oligos were ordered:

Oligo 1: GAT CCG ATG CCG TGG CTG ATG AGG CCG AAA GGC CGA AAC TGG TAG TT(SEQ ID NO. 1240)

Oligo 2: AAC TAC CAG TTT CGG CCT TTC GGC CTC ATC AGC CAC GGC ATC G (SEQID NO. 1241)

The oligos are phosphorylated individually in 1×kinase buffer(Gibco/BRL) and heat denatured and annealed by combining at 90° C. for10 min, then slow cooled to room temperature (22° C.). The vector wasprepared by digestion of pDAB 353 with Sst I and blunting the ends withT4 DNA polymerase. The vector was redigested with Bam HI and gelpurified as above. The annealed oligos are ligated to the vector in1×ligation buffer (Gibco/BRL) at 16° C. overnight. Potential clones weredigested with Bam HI/Eco RI and confirmed by sequencing.

Example 16 Plant Transformation Plasmids pDAB 367, Used in the Δ9Ribozyme Experiments, and pDAB353 Used in the GBSS Ribozyme Experiments

Part A pDAB367

Plasmid pDAB367 has the following DNA structure: beginning with the baseafter the final C residue of the Sph I site of pUC 19 (base 441; Ref.1), and reading on the strand contiguous to the LacZ gene coding strand,the linker sequence CTGCAGGCCGGCCTTAATTAAGCGGCCGCGTTTAAACGCCCGGGCATTTAAATGGCGCGCCGCGATCGCTTGCAGATCTGCATGGGTG (SEQ ID NO. 1242), nucleotides 7093 to 7344 ofCaMV DNA (2), the linker sequence CATCGATG, nucleotides 7093 to 7439 ofCaMV, the linker sequence GGGGACTCTAGAGGATCCAG (SEQ ID NO. 1243),nucleotides 167 to 186 of MSV (3), nucleotides 188 to 277 of MSV (3), aC residue followed by nucleotides 119 to 209 of maize Adh 1S containingparts of exon 1 and intron 1 (4), nucleotides 555 to 672 containingparts of Adh 1S intron 1 and exon 2 (4), the linker sequence GACGGATCTG(SEQ ID NO. 1244), and nucleotides 278 to 317 of MSV. This is followedby a modified BAR coding region from pIJ4104 (5) having the AGC serinecodon in the second position replaced by a GCC alanine codon, andnucleotide 546 of the coding region changed from G to A to eliminate aBgl II site. Next, the linker sequence TGAGATCTGAGCTCGAATTTCCCC (SEQ IDNO. 1245), nucleotides 1298 to 1554 of Nos (6), and a G residue followedby the rest of the pUC 19 sequence (including the Eco RI site).

PartB pDAB353

Plasmid pDAB353 has the following DNA structure: beginning with the baseafter the final C residue of the Sph I site of pUC 19 (base 441; Ref.1), and reading on the strand contiguous to the LacZ gene coding strand,the linker sequence CTGCAGATCTGCATGGGTG (SEQ ID NO. 1246), nucleotides7093 to 7344 of CaMV DNA (2), the linker sequence CATCGATG, nucleotides7093 to 7439 of CaMV, the linker sequence GGGGACTCTAGAG (SEQ ID NO.1247), nucleotides 119 to 209 of maize Adh 1S containing parts of exon 1and intron 1 (4), nucleotides 555 to 672 containing parts of Adh 1Sintron 1 and exon 2 (4), and the linker sequence GACGGATCCGTCGACC (SEQID NO. 1248), where GGATCC represents the recognition sequence for BamHI restriction enzyme. This is followed by the beta-glucuronidase (GUS)gene from pRAJ275 (7), cloned as an Nco I/Sac I fragment, the linkersequence GAATTTCCCC (SEQ ID NO. 1249), the poly A region in nucleotides1298 to 1554 of Nos (6), and a G residue followed by the rest of the pUC19 sequence (including the Eco RI site).

The following are herein incorporated by reference:

1. Messing, J. (1983) in “Methods in Enzymology” (Wu, R. et al., Eds)101:20-78.

2. Franck, A., H. Guilley, G. Jonard, K. Richards, and L. Hirth (1980)Nucleotide sequence of Cauliflower Mosaic Virus DNA. Cell 21:285-294.

3. Mullineaux, P. M., J. Donson, B. A. M. Morris-Krsinich, M. I.Boulton, and J. W. Davies (1984) The nucleotide sequence of Maize StreakVirus DNA. EMBO J. 3:3063-3068.

4. Dennis, E. S., W. L. Gerlach, A. J. Pryor, J. L. Bennetzen, A.Inglis, D. Llewellyn, M. M. Sachs, R. J. Ferl, and W. J. Peacock (1984)Molecular analysis of the alcohol dehydrogenase (Adh1) gene of maize.Nucl. Acids Res. 12:3983-4000.

5. White, J., S-Y Chang, M. J. Bibb, and M. J. Bibb (1990) A cassettecontaining the bar gene of Streptomyces hygroscopicus: a selectablemarker for plant transformation. Nucl. Acids. Res. 18:1062.

6. DePicker, A., S. Stachel, P. Dhaese, P. Zambryski, and H. M. Goodman(1982) Nopaline Synthase: Transcript mapping and DNA sequence. J. Molec.Appl. Genet. 1:561-573.

7. Jefferson, R. A. (1987) Assaying chimeric genes in plants: The GUSgene fusion system. Plant Molec. Biol. Reporter 5:387-405.

Example 17 Plasmid pDAB359 a Plant Transformation Plasmid which Containsthe Gamma-Zein Promoter, the Antisense GBSS, and a the NosPolyadenylation Sequence

Plasmid pDAB359 is a 6702 bp double-stranded, circular DNA that containsthe following sequence elements: nucleotides 1-404 from pUC18 whichinclude lac operon sequence from base 238 to base 404 and ends with theHindIII site of the M13mp18 polylinker (1,2); the Nos polyadenylationsequence from nucleotides 412 to 668 (3); a synthetic adapter sequencefrom nucleotides 679-690 which converts a Sac I site to an Xho I site bychanging GAGCTC to GAGCTT and adding CTCGAG; maize granule bound starchsynthase cDNA from bases 691 to 2953, corresponding to nucleotides1-2255 of SEQ. I.D. No. 25. The GBSS sequence in plasmid pDAB359 wasmodified from the original cDNA by the addition of a 5′ Xho I and a 3′Nco I site as well as the deletion of internal Nco I and Xho I sitesusing Klenow to fill in the enzyme recognition sequences. Bases 2971 to4453 are 5′ untranslated sequence of the maize 27 kD gamma-zein genecorresponding to nucleotides 1078 to 2565 of the published sequence (4).The gamma-zein sequence was modified to contain a 5′ Kpn I site and 3′BamH/SalI/Nco I sites. Additional changes in the gamma-zein sequencerelative to the published sequence include a T deletion at nucleotide104, a TACA deletion at nucleotide 613, a C to T conversion atnucleotide 812, an A deletion at nucleotide 1165 and an A insertion atnucleotide 1353. Finally, nucleotides 4454 to 6720 of pDAB359 areidentical to puc18 bases 456 to 2686 including the Kpn I/EcoRI/Sac Isites of the M13/mp18 polylinker from 4454 to 4471, a lac operonfragment from 4471 to 4697, and the β-lacatmase gene from 5642 to 6433(1, 2).

The following are incorporated by reference herein:

pUC18—Norrander, J., Kempe, T., Messing, J. Gene (1983) 26: 101-106;Pouwels, P. H., Enger-Valk, B. E., Brammar, W. J. Cloning Vectors,Elsevier 1985 and supplements

NosA—DePicker, A., Stachel, S., Dhaese, P., Zambryski, P., and Goodman,H. M. (1982) Nopaline Synthase: Transcript Mapping and DNA Sequence J.Molec. Appl. Genet. 1:561-573.

Maize 27 kD gamma-zein—Das, O. P., Poliak, E. L., Ward, K., Messing, J.Nucleic Acids Research 19, 3325-3330 (1991).

Example 18 Construction of Plasmid pDAB430, Containing Antisense Δ9Desaturase, Expressed by the Ubiquitin Promoter/intron (Ubil)

Part A Construction of plasmid pDAB421

Plasmid pDAB421 contains a unique blunt-end SrfI cloning site flanked bythe maize Ubiquitin promoter/intron and the nopaline synthasepolyadenylation sequences. pDAB421 was prepared as follows: digestion ofpDAB355 with restriction enzymes KpnI and BamHI drops out the R codingregion on a 2.2 kB fragment. Following gel purification, the vector wasligated to an adapter composed of two annealed oligonucleotides OF235and OF236. OF235 has the sequence 5′-GAT CCG CCC GGG GCC CGG GCG GTAC-3′ (SEQ ID NO. 1250) and OF236 has the sequence 5′-CGC CCG GGC CCC GGGCG-3′ (SEQ ID NO. 1251). Clones containing this adapter were identifiedby digestion and linearization of plasmid DNA with the enzymes SrfI andSmaI which cut in the adapter, but not elsewhere in the plasmid. Onerepresentative clone was sequenced to verify that only one adapter wasinserted into the plasmid. The resulting plasmid pDAB421 was used insubsequent construction of the Δ9 desaturase antisense plasmid pDAB430.

Part B Construction of plasmid pDAB430 (antisense Δ9 desaturase)

The antisense Δ9 desaturase construct present in plasmid pDAB430 wasproduced by cloning of an amplification product in the blunt-end cloningsite of plasmid pDAB421. Two constructs were produced simultaneouslyfrom the same experiment. The first construct contains the Δ9 desaturasegene in the sense orientation behind the ubiquitin promoter, and thec-myc tag fused to the C-terminus of the Δ9 desaturase open readingframe for immunological detection of overproduced protein in transgeniclines. This construct was intended for testing of ribozymes in a systemwhich did not express maize Δ9 desaturase. This construct was neverused, but the primers used to amplify and construct the Δ9 desaturaseantisense gene were the same. The Δ9 desaturase cDNA sequence describedherein was amplified with two primers. The N-terminal primer OF279 hasthe sequence 5′-GTG CCC ACA ATG GCG CTC CGC CTC AAC GAC-3′ (SEQ ID NO.1252). The underlined bases correspond to nucleotides 146-166 of thecDNA sequence. C-terminal primer OF280 has the sequence 5′-TCA TCA CAGGTC CTC CTC GCT GAT CAG CTT CTC CTC CAG TTG GAC CTG CCT ACC GTA-3′ (SEQID NO. 1253) and is the reverse complement of the sequence 5′-TAC GGTAGG GAC GTC CAA CTG GAG GAG AAG CTG ATC AGC GAG GAG GAC CTG TGA TGA-3′(SEQ ID NO. 1254). In this sequence the underlined bases correspond tonucleotides 1304-1324 of the cDNA sequence, the bases in italicscorrespond to the sequence of the c-myc epitope. The 1179 bp ofamplification product was purified through a 1.0% agarose gel, andligated into plasmid pDAB421 which was linearized with the restrictionenzyme Srf I. Colony hybridization was used to select clones containingthe pDAB421 plasmid with the insert. The orientation of the insert wasdetermined by restriction digestion of plasmid DNA with diagnosticenzymes KpnI and BamHI. A clone containing the Δ9 desaturase codingsequence in the sense orientation relative to the Ubiquitinpromoter/intron was recovered and was named pDAB429. An additional clonecontaining the Δ9 desaturase coding sequence in the anitsenseorientation relative to the promoter was named pDAB430. Plasmid pDAB430was subjected to sequence analysis and it was determined that thesequence contained three PCR induced errors compared to the expectedsequence. One error was found in the sequence corresponding to primerOF280 and two nucleotide changes were observed internal to the codingsequence. These errors were not corrected, because antisensedownregulation does not require 100% sequence identity between theantisense transcript and the downregulation target.

Example 19 Helium Blasting of Embryogenic Maize Cultures and theSubsequent Regeneration of Transgenic Progeny

Part A Establishment of embryogenic maize cultures. The tissue culturesemployed in transformation experiments were initiated from immaturezygotic embryos of the genotype “Hi-II”. Hi-II is a hybrid made byintermating 2 R₃ lines derived from a B73×A188 cross (Armstrong et al.1990). When cultured, this genotype produces callus tissue known as TypeII. Type II callus is friable, grows quickly, and exhibits the abilityto maintain a high level of embryogenic activity over an extended timeperiod.

Type II cultures were initiated from 1.5-3.0 mm immature embryosresulting from controlled pollinations of greenhouse grown Hi-II plants.The initiation medium used was N6 (Chu, 1978) which contained 1.0 mg/L2,4-D, 25 mM L-proline, 100 mg/L casein hydrolysate, 10 mg/L AgNO₃, 2.5g/L gelrite and 2% sucrose adjusted to pH 5.8. For approximately 2-8weeks, selection occurred for Type II callus and against nonembryogenicand/or Type I callus. Once Type II callus was selected, it wastransferred to a maintenance medium in which AgNO₃ was omitted andL-proline reduced to 6 mM.

After approximately 3 months of subculture in which the quantity andquality of embryogenic cultures was increased, the cultures were deemedacceptable for use in transformation experiments.

Part B Preparation of plasmid DNA. Plasmid DNA was adsorbed onto thesurface of gold particles prior to use in transformation experiments.The experiments for the GBSS target used gold particles which werespherical with diameters ranging from 1.5-3.0 microns (Aldrich ChemicalCo., Milwaukee, Wis.). Transformation experiments for the Δ9 desaturasetarget used 1.0 micron spherical gold particles (Bio-Rad, Hercules,Calif.). All gold particles were surface-sterilized with ethanol priorto use. Adsorption was accomplished by adding 74 μl of 2.5 M calciumchloride and 30 μl of 0.1 M spermidine to 300 μl of plasmid DNA andsterile H₂O. The concentration of plasmid DNA was 140 μg. The DNA-coatedgold particles were immediately vortexed and allowed to settle out ofsuspension. The resulting clear supernatent was removed and theparticles were resuspended in 1 ml of 100% ethanol. The final dilutionof the suspension ready for use in helium blasting was 7.5 mg DNA/goldper ml of ethanol.

Part C Preparation and helium blasting of tissue targets. Approximately600 mg of embryogenic callus tissue per target was spread over thesurface of petri plates containing Type II callus maintenance mediumplus 0.2 M sorbitol and 0.2 M mannitol as an osmoticum. After anapproximately 4 hour pretreatment, all tissue was transferred to petriplates containing 2% agar blasting medium (maintenance medium plusosmoticum plus 2% agar).

Helium blasting involved accelerating the suspended DNA-coated goldparticles towards and into prepared tissue targets. The device used wasan earlier prototype to the one described in a DowElanco U.S. Pat. No.5,141,131) which is incorporated herein by reference, although bothfunction in a similar manner. The device consisted of a high pressurehelium source, a syringe containing the DNA/gold suspension, and apneumatically-operated multipurpose valve which provided controlledlinkage between the helium source and a loop of pre-loaded DNA/goldsuspension.

Prior to blasting, tissue targets were covered with a sterile 104 micronstainless steel screen, which held the tissue in place during impact.Next, targets w ere placed under vacuum in the main chamber of thedevice. The DNA-coated gold particles were accelerated at the target 4times using a helium pressure of 1500 psi. Each blast delivered 20 μl ofDNA/gold suspension. Immediately post-blasting, the targets were placedback on maintenance medium plus osmoticum for a 16 to 24 hour recoveryperiod.

Part D Selection of transformed tissue and the regeneration of plantsfrom transgenic cultures. After 16 to 24 hours post-blasting, the tissuewas divided into small pieces and transferred to selection medium(maintenance medium plus 30 mg/L Basta™). Every 4 weeks for 3 months,the tissue pieces were non-selectively transferred to fresh selectionmedium. After 8 weeks and up to 24 weeks, any sectors foundproliferating against a background of growth inhibited tissue wereremoved and isolated. Putatively transformed tissue was subcultured ontofresh selection medium. Transgenic cultures were established after 1 to3 additional subcultures.

Once Basta™ resistant callus was established as a line, plantregeneration was initiated by transferring callus tissue to petri platecontaining cytokinin-based induction medium which were then placed inlow light (125 ft-candles) for one week followed by one week in highlight (325 ft-candles). The induction medium was composed of MS saltsand vitamins (Murashige and Skoog, 1962), 3 0 g/L sucrose, 100 mg/Lmyo-inositol, 5 mg/L 6-benzylaminopurine, 0.025 mg/L 2,4-D, 2.5 g/Lgelrite adjusted to pH 5.7. Following the two week induction period, thetissue was non-selectively transferred to hormone-free regenerationmedium and kept in high light. The regeneration medium was composed ofMS salts and vitamins, 30 g/L sucrose and 2.5 g/L gelrite adjusted to pH5.7. Both induction and regeneration media contained 30 mg/L Basta™.Tissue began differentiating shoots and roots in 2-4 weeks. Small (1.5-3cm) plantlets were removed and placed in tubes containing SH medium. SHmedium is composed of SH salts and vitamins (Schenk and Hildebrandt,1972), 10 g/L sucrose, 100 mg/L myo-inositol, 5 mL/L FeEDTA, and either7 g/L Agar or 2.5 g/L Gelrite adjusted to pH 5.8. Plantlets weretransferred to 10 cm pots containing approximately 0.1 kg of Metro-Mix®360 (The Scotts Co., Marysville, Ohio) in the greenhouse as soon as theyexhibited growth and developed a sufficient root system (1-2 weeks). Atthe 3-5 leaf stage, plants were trans ferred to 5 gallon pots containingapproximately 4 kg Metro-Mix® 360 and grown to maturity. These R₀ plantswere self-pollinated and/or cross-pollinated with non-transgenic inbredsto obtain transgenic progeny. In the case of transgenic plants producedfor the GBSS target, R₁ seed produced from R₀ pollinations wasreplanted. The R₁ plants were grown to maturity and pollinated toproduce R₂ seed in the quantities needed for the analyses.

Example 20 Production and Regeneration of Δ9 Transgenic Material

Part A Transformation and isolation of embryogenic callus. Six ribozymeconstructs, described previously, targeted to Δ9 desaturase weretransformed into regenerable Type II callus cultures as describedherein. These 6 constructs consisted of 3 active/inactive pairs; namely,RPA85/RPA113, RPA114/RPA115, and RPA118/RPA119. A total of 1621 tissuetargets were prepared, blasted, and placed into selection. From theseblasting experiments 334 independent Basta®-resistant transformationevents (“lines”) were isolated from selection. Approximately 50% ofthese lines were analyzed via DNA PCR or GC/FAME as a means ofdetermining which ones to move forward to regeneration and which ones todiscard. The remaining 50% were not analyzed either because they hadbecome non-embryogenic or contaminated.

Part B Regeneration of Δ9 plants from transgenic callus. Followinganalyses of the transgenic callus, twelve lines were chosen per ribozymeconstruct for regeneration, with 15 R₀ plants to be produced per line.These lines generally consisted of 10 analysis-positive lines plus 2negative controls, however, due to the poor regenerability of some ofthe cultures, plants were produced from less than 12 lines forconstructs RPA113, RPA115, RPA118, and RPA119. An overall total of 854R₀ plants were regenerated from 66 individual lines (see Table X). Whenthe plants reached maturity, self- or sib-pollinations were given thehighest priority, however, when this was not possible,cross-pollinations were made using the inbreds CQ806, CS716, OQ414, orHO₁ as pollen donors, and occasionally as pollen recipients. Over 715controlled pollinations have been made, with the majority (55%) beingcomprised of self- or sib-pollinations and the minority (45%) beingcomprised of F1 crosses. R₁ seed was collected approximately 45 dayspost-pollination.

Example 21 Production and Regeneration of Transgenic Maize for the GBSS

Part A Transformation of embryogenic maize callus and the subsequentselection and establishment of transgenic cultures. RPA63 and RPA64, anactive/inactive pair of ribozyme multimers targeted to GBSS, wereinserted along with bar selection plasinid pDAB308 into Type II callusas described herein. A total of 115 Basta™-resistant independenttransformation events were recovered from the selection of 590 blastedtissue targets. Southern analysis was performed on callus samples fromestablished cultures of all events to determine the status of the geneof interest.

Part B Regeneration of plants from cultures transformed with ribozymestargeted to GBSS as well as the advancement to the R₂ generation. Plantswere regenerated from Southern “positive” transgenic cultures and grownto maturity in a greenhouse. The primary regenerates were pollinated toproduce R₁ seed. From 30 to 45 days after pollination, seed washarvested, dried to the correct moisture content, and replanted. A totalof 752 R₁ plants, representing 16 original lines, were grown to sexualmaturity and pollinated. Approximately 19 to 22 days after pollination,ears were harvested and 30 kernels were randomly excised per ear andfrozen for later analyses.

Example 22 Testing of GBSS-Targeted Ribozymes in Maize Black MexicanSweet (BMS) Stably Transformed Callus

Part A Production of BMS callus stably transformed with GBSS andGBSS-targeted ribozymes. BMS does not produce a GBSS mRNA which ishomologous to that found endogenously in maize. Therefore, a doubletransformation system was developed to produce transformants whichexpressed both target and ribozymes. “ZM” BMS suspensions (obtained fromJack Widholm, University of Illinois, also see W. F. Sheridan, “BlackMexican Sweet Corn: Its Use for Tissue Cultures” in Maize for BiologicalResearch, W. F. Sheridan, editor. University Press. University of NorthDakokta, Grand Forks, N. Dak., 1982, pp. 385-388) were prepared forhelium blasting four days after subculture by transfer to a 100×20 mmPetri plate (Fisher Scientific, Pittsburgh, Pa.) and partial removal ofliquid medium, forming a thin paste of cells. Targets consisted of100-125 mg fresh weight of cells on a ½″ antibiotic disc (Schleicher andSchuell, Keene, N.H.) placed on blasting medium, DN6 [N6 salts andvitamins (Chu et al., 1978), 20 g/L sucrose, 1.5 mg/L2,4-dichlorophenoxyacetic acid (2,4-D), 25 mM L-proline; pH=5.8 beforeautoclaving 20 minutes at 121° C.] solidified with 2% TC agar (JRHBiosciences, Lenexa, Kas.) in 60×20 mm plates. DNA was precipitated ontogold particles. For the first transformation, pDAB 426 (Ubi/GBSS) andpDAB 308 (35T/Bar) were used. Targets were individually shot usingDowElanco Helium Blasting Device I. With a vacuum pressure of 650 mm Hgand at a distance of 15.5 cm from target to device nozzle, each samplewas blasted once with DNA/gold mixture at 500 psi. Immediately afterblasting, the antibiotic discs were transferred to DN6 medium made with0.8% TC agar for one week of target tissue recovery. After recovery,each target was spread onto a 5.5 cm Whatman #4 filter placed on DN6medium minus proline with 3 mg/L Basta® (Hoechst, Frankfort, Germany).Two weeks later, the filters were transferred to fresh selection mediumwith 6 mg/L Basta®. Subsequent transfers were done at two weekintervals. Isolates were picked from the filters and placed on AMCF-ARMmedium (N6 salts and vitamins, 20 g/L sucrose, 30 g/L mannitol, 100 mg/Lacid casein hydrolysate, and 1 mg/L 2,4-D, 24 mM L-proline; pH=5.8before autoclaving 20 minutes at 121° C.) solidified with 0.8% TC agarcontaining 6 mg/L Basta®. Isolates were maintained by subculture tofresh medium every two weeks.

Basta®-resistant isolates which expressed GBSS were subjected to asecond transformation. As with BMS suspensions, targets of transgeniccallus were prepared 4 days after subculture by spreading tissue onto ½″filters. However, AMCF-ARM with 2% TC agar was used for blasting, due tomaintenance of transformants on AMCF-ARM selection media. Each samplewas covered with a sterile 104 μm mesh screen and blasting was done at1500 psi. Target tissue was co-bombarded with pDAB 319 (35S-ALS;35T-GUS) and RPA63 (active ribozyme multimer) or pDAB3 19 and RPA64(inactive ribozyme multimer), or shot with pDAB 319 alone. Immediatelyafter blasting, all targets were transferred to nonselective medium(AMCF-ARM) for one week of recovery. Subsequently, the targets wereplaced on AMCF-ARM medium containing two selection agents, 6 mg/L Basta®and 2 μg/L chlorsulfuron (CSN). The level of CSN was increased to 4 ug/Lafter 2 weeks. Continued transfer of the filters and generation ofisolates was done as described in the first transformation, withisolates being maintained on AMCF-ARM medium containing 6 mg/L Basta and4 μg/L CSN.

Part B Analysis of BMS stable transformants expressing GBSS andGBSS-targeted ribozymes. Isolates from the first transformation wereevaluated by Northern blot analysis for detection of a functional targetgene (GBSS) and to detennine relative levels of expression. In 12 of 25isolates analyzed, GBSS transcript was detected. A range of expressionwas observed, indicating an independence of transfornation events.Isolates generated from the second transformation were evaluated byNorthern blot analysis for detection of continued GBSS expression and byRT-PCR to screen for the presence of ribozyme transcript. Of 19 isolatestested from one previously transformed line, 18 expressed the activeribozyme, RPA63, and all expressed GBSS. GBSS was detected in each of 6vector controls; ribozyme was not expressed in these samples. Asdescribed herein, RNase protection assay (RPA) and Northern blotanalysis were performed on ribozyme-expressing and vector controltissues to compare levels of GBSS transcript in the presence or absenceof active ribozyme. GBSS values were normalized to an internal control(Δ9 desaturase); Northern blot data is shown in FIG. (25). Northern blotresults revealed a significantly lower level of GBSS message in thepresence of ribozyme, as compared to vector controls. RPA data showedthat some of the individual samples expressing active ribozyme (“L” and“O”) were significantly different from vector controls and similar to anontransformed control.

Example 23 Analysis of Plant and Callus Materials

Plant material co-transformed with the pDAB308 and one of the followingribozyme containing vectors, pRPA63, pRPA64, pRPA85, pRPA113, pRPA114,pRPA115, pRPA118 or pRPA119 were analyzed at the callus level, Ro leveland select lines analyzed at the F1 level. Leaf material was harvestedwhen the plantlets reached the 6-8 leaf stage. DNA from the plant andcallus material was prepared from lyophilized tissue as described bySaghai-Maroof et al.(supra). Eight micrograms of each DNA was digestedwith the restriction enzymes specific for each construct usingconditions suggested by the manufacturer (Bethesda Research Laboratory,Gaithersburg, Md.) and separated by agarose gel electrophoresis. The DNAwas blotted onto nylon membrane as described by Southern, E. 1975“Detection of specific sequences among DNA fragments separated by gelelectrophoresis,” J Mol. Biol. 98:503 and Southern, E. 1980 “Gelelectrophoresis of restriction fragments” Methods Enzmol. 69:152, whichare incorporated by reference herein.

Probes specific for the ribozyme coding region were hybridized to themembranes. Probe DNA was prepared by boiling 50 ng of probe DNA for 10minutes then quick cooling on ice before being added to the Ready-To-GoDNA labeling beads (Pharmacia LKB, Piscataway, N.J.) with 50 microcuriesof α³²P-dCTP (Amersham Life Science, Arlington Heights, Ill.). Probeswere hybridized to the genomic DNA on the nylon membranes. The membraneswere washed at 60° C. in 0.25×SSC and 0.2% SDS for 45 minutes, blotteddry and exposed to XAR-5 film overnight with two intensifying screens.

The DNA from the RPA63 and RPA64 was digested with the restrictionenzymes HindIII and EcoRI and the blots containing these samples werehybridized to the RPA63 probe. The RPA63 probe consists of the RPA63ribozyme multimer coding region and should produce a single 1.3 kbhybridization product when hybridized to the RPA63 or RPA64 materials.The 1.3 kb hybridization product should contain the enhanced 35Spromoter, the AdhI intron, the ribozyme coding region and the nopalinesynthase poly A 3′ end. The DNA from the RPA85 and RPA113 was digestedwith the restriction enzymes HindIII and EcoRI and the blots containingthese samples were hybridized to the RPA122 probe. RPA122 is the 252multimer ribozyme in pDAB 353 replacing the GUS reporter. The RPA122probe consists of the RPA122 ribozyme multimer coding region and thenopaline synthase 3′ end and should produce a single 2.1 kbhybridization product when hybridized to the RPA85 or RPA113 materials.The 2.1 kb hybridization product should contain the enhanced 35Spromoter, the AdhI intron, the bar gene, the ribozyme coding region andthe nopaline synthase poly A 3′ end. The DNA from the RPA114 and RPA115was digested with the restriction enzymes HindIII and SmaI and the blotscontaining these samples were hybridized to the RPA115 probe. The RPA115probe consist of the RPA115 ribozyme coding region and should produce asingle 1.2 kb hybridization product when hybridized to the RPA114 orRPA115 materials. The 1.2 kb hybridization product should contain theenhanced 35S promoter, the AdhI intron, the ribozyme coding region andthe nopaline synthase poly A 3′ end. The DNA from the RPA118 and RPA119was digested with the restriction enzymes HindIII and SmaI and the blotscontaining these samples were hybridized to the RPA118 probe. The RPA118probe consist of the RPA118 ribozyme coding region and should produce asingle 1.3 kb hybridization product when hybridized to the RPA118 orRPA119 materials. The 1.3 kb hybridization product should contain theenhanced 35S promoter, the Adhl intron, the ribozyme coding region andthe nopaline synthase poly A 3′ end.

Example 24 Extraction of Genomic DNA from Transgenic Callus

Three hundred mg of actively growing callus were quick frozen on dryice. It was ground to a fine powder with a chilled Bessman TissuePulverizer (Spectrum, Houston, Tex.) and extracted with 400 μl of 2×CTABbuffer (2% Hexadecyltrimethylammonium Bromide, 100 mM Tris pH 8.0, 20 mMEDTA, 1.4 M NaCl, 1% polyvinylpyrrolidone). The suspension was lysed at65° C. for 25 minutes, then extracted with an equal volume ofchloroform:isoamyl alcohol. To the aqueous phase was added 0.1 volumesof 10% CTAB buffer (10% Hexadecyltrimethylammonium Bromide, 0.7 M NaCl).Following extraction with an equal volume of chloroform:isoamyl alcohol,0.6 volumes of cold isopropyl alcohol was added to the aqueous phase,and placed at −20° C. for 30 minutes. After a 5 minute centrifugation at14,000 rpm, the resulting precipitant was dried for 10 minutes undervacuum. It was resuspended in 200 μl TE (10 mM Tris, 1 mM EDTA, pH 8.0)at 65° C. for 20 minutes. 20% Chelex (Biorad,) was added to the DNA to afinal concentration of 5% and incubated at 56° C. for 15-30 minutes toremove impurities. The DNA concentration was measured on a HoeferFluorimeter (Hoefer, San Francisco).

Example 25 PCR Analysis of Genomic Callus DNA

Use of Polymerase Chain Reaction (PCR) to demonstrate the stableinsertion of ribozyme genes into the chromosome of transgenic maizecalli.

Part A Method used to detect ribozyme DNA

The Polymerase Chain Reaction (PCR) was performed as described in thesuppliers protocol using AmpliTaq DNA Polymerase (GeneAmp PCR kit,Perkin Elmer, Cetus). Aliquots of 300 ng of genomic callus DNA, 1 μl ofa 50 μM downstream primer (5′ CGC AAG ACC GGC AAC AGG 3′; SEQ ID NO.1255), 1 μl of an upstream primer and 1 μl of Perfect Match (Stratagene,Calif.) PCR enhancer were mixed with the components of the kit. The PCRreaction was performed for 40 cycles using the following parameters;denaturation at 94° C. for 1 minute, annealing at 55° C. for 2 minutes,and extension at 72° C. for 3 mins. An aliquot of 0.2×vol. of each PCRreaction was electrophoresised on a 2% 3:1 Agarose (FMC) gel usingstandard TAE agarose gel conditions.

Part B Upstream primer used for detection of Δ9 desaturase ribozymegenes

RPA85/RPA113 251 multimer fused to BAR 3′ ORF

RPA114/RPA115 258 ribozyme monomer

RPA118/RPA119 452 ribozyme multimer 5′TGG ATT GAT GTG ATA TCT CCA C 3′(SEQ ID NO. 1256) This primer is used to amplify across the Eco RV sitein the 35S promoter. Primers were prepared using standard oligosynthesis protocols on an Applied Biosystems Model 394 DNA/RNAsynthesizer.

Example 26 Preparation of Total RNA from Transgenic Maize Calli andPlant

Part A Preparation of total RNA from transgenic non-regenerable andregenerable callus tissue. Three hundred milligrams of actively growingcallus was quick frozen on dry ice. The tissue was ground to a finepowder with a chilled Bessman Tissue Pulverizer (Spectrum, Houston,Tex.) and extracted with RNA Extraction Buffer (50 mM Tris-HCl pH 8.0,4% para-amino salicylic acid, 1% Tri-iso-propylnapthalenesulfonic acid,10 mM dithiothreitol, and 10 mM Sodium meta-bisulfite) by vigorousvortexing. The homogenate was then extracted with an equal volume ofphenol containing 0.1% 8-hydroxyquinoline. After centrifugation, theaqueous layer was extracted with an equal volume of phenol containingchloroform:isoamyl alcohol (24:1), followed by extraction withchloroform:octanol (24:1). Subsequently, 7.5 M Ammonium acetate wasadded to a final concentration of 2.5 M, the RNA was precipitated for 1to 3 hours at 4° C. Following 4° C. centrifugation at 14,000 rpm, RNAwas resuspended in sterile water, precipitated with 2.5 M NH₄OAc and 2volumes of 100% ethanol and incubated ovemite at −20° C. The harvestedRNA pellet was washed with 70% ethanol and dried under vacuum. RNA wasresuspended in sterile H₂O and stored at −80° C.

Part B Preparation of total RNA from transgenic maize plants. A five cmsection (˜150 mg) of actively growing maize leaf tissue was excised andquick frozen in dry ice. The leaf was ground to a fine powder in achilled mortar. Following manufactorers instructions, total RNA waspurified from the powder using a Qaigen RNeasy Plant Total RNA kit(Qiagen Inc., Chatsworth, Calif.). Total RNA was released from theRNeasy columns by two sequential elution spins of prewarmed (50° C.)sterile water (30 μl each) and stored at −80° C.

Example 27 Use of RT-PCR Analysis to Demonstrate Expression of RibozymeRNA in Transgenic Maize Calli and Plants

Part A Method used to detect ribozyme RNA. The ReverseTranscription-Polyinerase Chain Reaction (RT-PCR) was performed asdescribed in the suppliers protocol using a thermostable rTth DNAPolymerase (rTth DNA Polymerase RNA PCR kit, Perkin Elmer Cetus).Aliquots of 300 ng of total RNA (leaf or callus) and 1 μl of a 15 μMdownstream primer (5′ CGC AAG ACC GGC AAC AGG 3′; SEQ ID NO. 1257) weremixed with the RT components of the kit. The reverse transcriptionreaction was performed in a 3 step ramp up with 5 minute incubations at60° C., 65° C., and 70° C. For the PCR reaction, 1 μl of upstream primerspecific for the ribozyme RNA being analyzed was added to the RTreaction with the PCR components. The PCR reaction was performed for 35cycles using the following parameters; incubation at 96° C. for 1minute, denaturation at 94° C. for 30 seconds, annealing at 50° C. for30 seconds, and extension at 72° C. for 3 mins. An aliquot of 0.2×vol.of each RT-PCR reaction was electrophoresed on a 2% 3:1 Agarose (FMC)gel using standard TAE agarose gel conditions.

Part B Specific upstream primers used for detection of GBSS ribozymes.

GBSS Active and Inactive Multimer

5′ CAG ATC AAG TGC AAA GCT GCG GAC GGA TCT G 3′ (SEQ ID NO. 1258). Thisprimer covers the Adh I intron footprint upstream of the first ribozymearm. GBSS 918 Intron (−) Monomer:

5′ ATC CGA TGC CGT GGC TGA TG 3′ (SEQ ID NO. 1259). This primer coversthe 10 base pair ribozyme arm and the first 6 bases of the ribozymecatalytic domain. GBSS ribozyrne expression in transgenic callus andplants was confirmed by RT-PCR.

GBSS multimer ribozyme expression in stably transformed callus was alsodetermined by Ribonuclease Protection Assay.

Part C Specific upstream primers used for detection of Δ9 desaturaseribozymes.

RPA85/RPA113 252 multimer fused to BAR 3′ ORF

5′ GAT GAG ATC CGG TGG CAT TG 3′ (SEQ ID NO. 1260)

This primer spans the junction of the BAR gene and the RPA85/113ribozyme. RPA114/RPA115 259 ribozyme monomer

5′ ATC CCC TTG GTG GAC TGA TG 3′ (SEQ ID NO. 1261)

This primer covers the 10 base pair ribozyme arm and the first 6 basesof the ribozyme catalytic domain. RPA118/RPA119 453 ribozyme multimer

5′ CAG ATC AAG TGC AAA GCT GCG GAC GGA TCT G 3′ (SEQ ID NO. 1262)

This primer covers the Adh I intron footprint upstream of the firstribozyme arm. Expression of Δ9 desaturase ribozymes in transgenic plantlines 85-06, 113-06 and 85-15 were confirmed by RT-PCR.

Primers were prepared using standard oligo synthesis protocols on anApplied Biosystems Model 394 DNA/RNA synthesizer.

Example 28 Demonstration of Ribozyme Mediated Reduction in Target mRNALevels in Transgenic Maize Callus and Plants

Part A Northern analysis method which was used to demonstratedreductions in target mRNA levels. Five μg of total RNA was dried undervacuum, resuspended in loading buffer (20 mM phosphate buffer pH 6.8, 5mM EDTA; 50% formamide: 16% formaldehyde: 10% glycerol) and denaturedfor 10 minutes at 65° C. Electrophoresis was at 50 volts through 1%agarose gel in 20 mM phosphate buffer (pH 6.8) with bufferrecirculation. BRL 0.24-9.5 Kb RNA ladder (Gibco/BRL, Gaithersburg, Md.)were stained in gels with ethiduim bromide. RNA was transferred toGeneScreen membrane filter (DuPont NEN, Boston Mass.) by capillarytransfer with sterile water. Hybridization was performed as described byDeLeon et al. (1983) at 42° C., the filters were washed at 55° C. toremove non-hybridized probe. The blot was probed sequentially with cDNAfragments from the target gene and an internal RNA control gene. Theinternal RNA standard was utilized to distinguish variation in targetmRNA levels due to loading or handling errors from true ribozymemediated RNA reductions. For each sample the level of target mRNA wascompared to the level of control mRNA within that sample. Fragments werepurified by Qiaex resin (Qaigen Inc. Chatsworth, Calif.) from 1× TAEagarose gels. They were nick-translated using an Amersham NickTranslation Kit (Amersham Corporation, Arlington Heights, Ill.) withalpha ₃₂P dCTP. Autoradiography was at −70° C. with intensifying screens(DuPont, Wilmington Del.) for one to three days. Autoradiogram signalsfor each probe were measured after a 24 hour exposure by densitometerand a ratio of target/internal control mRNA levels was calculated.

Ribonuclease protection assays were performed as follows: RNA wasprepared using the Qiagen RNeasy Plant Total RNA Kit from either BMSprotoplasts or callus material. The probes were made using the AmbionMaxiscript kit and were typically 10⁸ cpm/microgram or higher. Theprobes were made the same day they were used. They were gel purified,resuspended in RNase-free 10 mM Tris (pH 8) and kept on ice. Probes werediluted to 5×10⁵ cpm/ul immediately before use. 5 μg of RNA derived fromcallus or 20 μg of RNA derived from protoplasts was incubated with 5×10⁵cpm of probe in 4M Guanidine Buffer. [4M Guanidine Buffer: 4M GuanidineThiocyanate/0.5% Sarcosyl/25 mM Sodium Citrate (pH 7.4)]. 40 ul of PCRmineral oil was added to each tube to prevent evaporation. The sampleswere heated to 95° for 3 minutes and placed immediately into a 45° waterbath. Incubation continued overnight. 600 μl of RNase Treatment Mix wasadded per sample and incubated for 30 minutes at 37° C. (RNase TreatmentMix: 400 mM NaCl, 40 units/ml RNase A and T1). 12 μl of 20% SDS wereadded per tube, immediately followed by addition of 12 ul (20 mg/ml)Proteinase K to each tube. The tubes were vortexed gently and incubatedfor 30 minutes at 37° C. 750 ul of room temperature RNase-freeisopropanol was added to each tube, and mixed by inverting repeatedly toget the SDS into solution. The samples were then microfuged at top speedat room temperature for 20 minutes. The pellets were air dried for 45minutes. 15 ul of RNA Running Buffer was added to each tube, andvortexed hard for 30 seconds. (RNA Running Buffer: 95% Formamide/20 mMEDTA/0.1% Bromophenol Blue/0.1% Xylene Cyanol). The sample was heated to95° C. for 3 minutes, and loaded onto an 8% denaturing acrylamide gel.The gel was vacuum dried and exposed to a phosphorimager screens for 4to 12 hours.

Part B Results demonstrating reductions in GBSS mRNA levels innongenerable callus expressing both a GBSS and GBSS targeted ribozymeRNA. The production of nonregenerable callus expressing RNAs for theGBSS target gene and an active multimer ribozyme targeted to GBSS mRNAwas performed. Also produced were transgenics expressing GBSS and aribozyme (−) control RNA. Total RNA was prepared from the transgeniclines. Northern analysis was performed on 7 ribozyme (−) controltransformants and 8 active RPA63 lines. Probes for this analysis were afull length maize GBSS cDNA and a maize Δ9 cDNA fragment. To distinguishvariation in GBSS mRNA levels due to loading or handling errors fromtrue ribozyme mediated RNA reductions, the level of GBSS mRNA wascompared to the level of Δ9 mRNA within that sample. The level of fulllength GBSS transcript was compared between ribozyme expressing andribozyme minus calli to identify lines with ribozyme mediated target RNAreductions. Blot to blot variation was controlled by performingduplicate analyses.

A range in GBSS/Δ9 ratio was observed between ribozyme (−) transgenics.The target mRNA is produced by a transgene and may be subject to morevariation in expression then the endogenous Δ9 mRNA. Active lines (RPA63) AA, EE, KK, and JJ were shown to reduce the level of GBSS/Δ9 mostsignificantly, as much as 10 fold as compared to ribozyme (−) controltransgenics this is graphed in FIG. 25. Those active lines were shown tobe expressing GBSS targeted ribozyme by RT-PCR as described herein.

Reductions in GBSS mRNA compared to Δ9 mRNA were also seen by RNAseprotection assay.

Part C Demonstration of reductions in Δ9 desaturasc levels in transgenicplants expressing ribozymes targeted to Δ9 desaturase mRNA. The highstearate transgenics, RPA85-06 and RPA85-15, each contained an intactcopy of the fused ribozyme multimer gene. Within each line, plants werescreened by RT-PCR for the presence of ribozyme RNA. Using the protocoldescribed in Example 27. RPA85 ribozyme expression was demonstrated inplants of the 85-06 and 85-15 lines which contained high stearic acid intheir leaves. Northern analysis was performed on the six high stearateplants from each line as well as non-transformed (NT) and transformedcontrol (TC) plants. The probes for this analysis were cDNA fragmentsfrom a maize Δ9 desaturase cDNA and a maize actin cDNA. To distinguishvariation in Δ9 mRNA levels due to loading or handling errors from trueribozyme mediated RNA reductions, the level of Δ9 mRNA was compared tothe level of actin mRNA within that sample. Using densitometer readingsdescribed above a ratio was calculated for each sample. Δ9/actin ratiovalues ranging from 0.55 to 0.88 were calculated for the 85-06 plants.The average Δ9/actin value for non-transformed controls was 2.7. Thereis an apparent 4 fold reduction in Δ9/actin ratios between 85-06 and NTleaves. Comparing Δ9/actin values between 85-06 high stearate and TCplants, on average a 3 fold reduction in Δ9/actin was observed for the85-06 plants. This data is graphed in FIG. 26. Ranges in Δ9/actin ratiosfrom 0.35 to 0.53, with an average of 0.43 were calculated for theRPA85-15 high stearate transgenics. In this experiment the averageΔ9/actin ratio for the NT plants was 1.7. Comparing the average Δ9/actinratio between NT controls and 85-15 high stearate plants, a 3.9 foldreduction in 85-15 Δ9 mRNA was demonstrated. An apparent 3 foldreduction in Δ9 mRNA level was observed for RPA85-15 high stearatetransgenics when Δ9/actin ratios were compared between 85-15 highstearate and normal stearate (TC) plants. These data are graphed in FIG.27. These data indicate ribozyme-mediated reduction of Δ9-desaturasemRNA in transgenic plants expressing RPA85 ribozyme, and producingincreased levels of stearic acid in the leaves.

Example 29 Evidence of Δ9 Desaturase Down Regulation in Maize Leaves asa Result of Active Ribozyme Activity

Plants were produced which were transformed with inactive versions ofthe Δ9 desaturase ribozyme genes. Data was presented demonstratingcontrol levels of leaf stearate in the inactive Δ9 ribozyme transgeniclines RPA113-06 and 113-17. Ribozyme expression and northern analysiswas performed for the RPA113-06 line. Δ9 desaturase protein levels weredetermined in plants of the RPA113-17 line. Ribozyme expression wasmeasured as described herein. Plants 113-06-04, -07, and -10 expresseddetectable levels of RPA 113 inactive Δ9 ribozyme. Northern analysis wasperformed on 5 plants of the 113-06 line with leaf stearate ranging from1.8-3.9%, all of which fall within the range of controls. No reductionin Δ9 desaturase mRNA correlating with ribozyme expression or elevationsin leaf stearate were found in the RPA113-06 plants as compared tocontrols, graphed in FIG. 28. Protein analysis did not indicate anyreduction in Δ9 desaturase protein levels correlating with elevated leafstearate in the RPA113-17 plants. This data is graphed in FIG. 29(a).Taken together, the data from the two RPA113 inactive transgenic linesindicate ribozyme activity is responsible for the high strearatephenotype observed in the RPA85 lines. The RPA85 ribozyme is the activeversion of the RPA113 ribozyme.

Example 30 Demonstration of Ribozyme Mediated Reduction in Stearoyl-ACPΔ9 Desaturase Levels in Maize Leaves (RO) Δ9 Desaturase Levels in MaizeLeaves (R0)

Part A Partial purification of stearoyl-ACP Δ9-desaturase from maizeleaves. All procedures were performed at 4° C. unless stated otherwise.Maize leaves (50 mg) were harvested and ground to a fine powder inliquid N₂ with a mortar and pestle. Proteins were extracted in one equalvolume of Buffer A consisting of 25 mM sodium-phosphate pH 6.5, 1 mMethylenediaminetetraacetic acid, 2 mM dithiothreitol, 10 mMphenylmethylsulfonyl fluoride, 5 mM leupeptin, and 5 mM antipapin. Thecrude homogenate was centrifuged for 5 minutes at 10,000×g. Thesupernatant was assayed for total protein concentration by Bio-Radprotein assay kit (Bio-Rad Laboratories, Hercules, Calif.). One hundredmicrograms of total protein was brought up to a final volume of 500 μlin Buffer A, added to 50 μl of mixed SP-sepharose beads (PharmaciaBiotech Inc., Piscataway, N.J.), and resuspended by vortexing briefly.Proteins were allowed to bind to sepharose. beads for 10 minutes whileon ice. After binding, the Δ9 desaturase-sepharose material wascentrifuged (10,000×g) for 10 seconds, decanted, washed three times withBuffer A (500 μl), and washed one time with 200 mM sodium chloride (500μl). Proteins were eluted by boiling in 50 μl of Treatment buffer (125mM Tris-Cl pH 6.8, 4% sodium dodecyl sulfate, 20% glycerol, and 10%2-mercaptoethanol) for 5 mintues. Samples were centrifuged (10,000 ×g)for 5 minutes. The supernatant was saved for Western anaylsis and thepellet consisting of sepharose beads was discarded.

Part B Western analysis method which was used to demonstrate reductionsin stearoyl-ACP Δ9 desaturase. Partially purified proteins wereseparated on sodium dodecyl sulfate (SDS)-polyacrylamide gels (10% PAGE)as described by Laemmli, U.K. (1970) Cleavage of structural proteinsduring assembly of the head of phage T4, Nature 227, 660-685. Todistinguish variation in Δ9 desaturase levels, included on each blot asa reference was purified and quantified overexpressed Δ9 desaturase fromE. coli as described hereforth. Proteins were electrophoreticallytransferred to ECL™ nitrocellulose membranes (Amersham Life Sciences,Arlington Heights, Ill.) using a Phannacia Semi-Dry Blotter (PharmaciaBiotech Inc., Piscataway, N.J.), using Towbin buffer (Towbin et al.1979). The nonspecific binding sites were blocked with 10% dry milk inphosphate buffer saline for 1 h. Immunoreactive polypeptides weredetected using the ECL™ Western Blotting Detection Reagent (AmershamLife Sciences, Arlington Heights, Ill.) with rabbit antiserum raisedagainst E. coli expressed maize Δ9 desaturase. The antibody was producedaccording to standard protocols by Berkeley Antibody Co. The secondaryantibody was goat antirabbit serum conjugated to horseradish peroxidase(BioRad). Autoradiograms were scanned with a densitometer and quantifiedbased on the relative amount of purified E. coli Δ9 desaturase. Theseexperiments were duplicated and the mean reduction was recorded.

Part C Demonstration of Reductions in Δ9 desaturase levels in R0 maizeleaves expressing ribozymes targeted to Δ9 desaturase mRNA. The highstearate transgenic line, RPA85-15, contains an intact copy of the fusedmultimer gene. Δ9 desaturase was partially purified from R0 maizeleaves, using the protocol described herein. Western analysis wasperformed on ribozyme active (RPA85-15) and ribozyme inactive(RPA113-17) plants and nontransformed (HiII) plants as described abovein part B. The natural variation of Δ9 desaturase was determined for thenontransformed line (HiII) by Western analysis see FIG. 29A. Noreduction in Δ9 desaturase was observed with the ribozyme inactive lineRPA113-17, all of which fell within the range as compared to thenontransformed line (HiII). An apparent 50% reduction of Δ9 desaturasewas observed in six plants of line RPA85-15 (FIG. 29B) as compared withthe controls. Concurrent with this, these same six plants also hadincreased stearate and reduced Δ9 desaturase mRNA (As described inExamples 28 and 32). However, nine active ribozyme plants from lineRPA85-15 did not have any significant reduction as compared withnontransformed line (HiII) and inactive ribozyme line (RPA113-17) (FIGS.29A and B). Collectively, these results suggest that the ribozymeactivity in the six plants from line RPA85-15 is responsible for thereduced Δ9 desaturase.

Example 31 E. coli Expression and Purification of Maize Δ-9 DesaturaseEnzyme

Part A The mature protein encoding portion of the maize Δ-9 desaturasecDNA was inserted into the bacterial T7 expression vector pET9D (NovagenInc., Madison, Wis.). The mature protein encoding region was deducedfrom the mature castor bean polypeptide sequence. The alanine atposition 32 (nts 239-241 of cDNA) was designated as the first residue.This is found within the sequence Ala.Val.Ala.Ser.Met.Thr. Restrictionendonuclease Nhe I site was engineered into the maize sequence by PCR,modifying GCCTCC to GCTAGC and a BamHI site was added at the 3′ end.This does not change the amino acid sequence of the protein. The cDNAsequence was cloned into pET9d vector using the Nhe I and Bam HI sites.The recombinant plasmid is designated as pDAB428. The maize Δ-9desaturase protein expressed in bacteria has an additional methionineresidue at the 5′ end. This pDAB428 plasmid was transformed into thebacterial strain BL21 (Novagen, Inc., Madison, Wis.) and plated onLB/kanamycin plates (25 mg/ml). Colonies were resuspended in 10 ml LBwith kanamycin (25 mg/ml) and IPTG (1 mM) and were grown in a shaker for3 hours at 37° C. The cells were harvested by centrifugation at 1000×gat 4° C. for 10 minutes. The cells were lysed by freezing and thawingthe cell pellet 2×, followed by the addition of 1 ml lysis buffer (10 mMTris-HCl pH 8.0, 1 mM EDTA, 150 mM NaCl, 0.1% Triton X100, 100 ug/milDNAse I, 100 ug/mI RNAse A, and 1 mg/ml lysozyme). The mixture wasincubated for 15 minutes at 37° C. and then centrifuged at 1000×g for 10minutes at 4° C. The supernatant is used as the soluble proteinfraction.

The supernatant, adjusted to 25 mM sodium phosphate buffer (pH 6.0), waschilled on ice for 1 hr. Afterwards, the resulting flocculantprecipitant was removed by centrifugation. The ice incubation step wasrepeated twice more after which the solution remained clear. Theclarified solution was loaded onto a Mono S HR 10/10 column (Pharmacia)that had been equilibrated in 25 mM sodium phosphate buffer (pH 6.0).Basic proteins bound to the column matrix were eluted using a 0-500 mMNaCl gradient over 1 hr (2 ml/min; 2 ml fractions). The putative proteinof interest was subjected to SDS-PAGE, blotted onto PVDF membrane,visualized with coomassie blue, excised, and sent to Harvard Microchemfor amino-terminal sequence analysis. Comparison of the protein's aminoterminal sequence to that encoded by the cDNA clone revealed that theprotein was indeed Δ9. Spectrophotometric analysis of the diiron-oxocomponent associated with the expressed protein (Fox et al., 1993 Proc.Natl. Acad. Sci. USA. 90, 2486-2490), as well as identification using aspecific nonheme iron stain (Leong et al., 1992 Anal. Biochem. 207,317-320) confirmed that the purified protein was Δ-9.

Part B Production of polyclonal antiserum

The E. coli produced Δ-9 protein, as determined by amino terminalsequencing, was gel purified via SDS-PAGE, excised, and sent in the gelmatrix to Berkeley Antibody Co., Richmond, Calif., for production ofpolyclonal sera in rabbits. Titers of the antibodies against Δ-9 wereperformed via western analysis using the ECL Detection system (Amersham,Inc.)

Part C Purification of Δ9 desaturase from corn kernels

Protein Precipitation: Δ9 was purified from corn kernels followinghomogenization using a Warring blender in 25 mM sodium phosphate buffer(pH 7.0) containing 25 mM sodium bisulfite and a 2.5%polyvinylpolypyrrolidone. The crude homogenate was filtered throughcheesecloth, centrifuged (10,000×g) for 0.25 h and the resultingsupernatant was filtered once more through cheesecloth. In some cases,the supernatant was fractionated via saturated ammonium sulfateprecipitation by precipitation at 20% v/v followed by 80% v/v. Extractsobtained from high oil germplasm were fractionated by adding a 50%polyethylene glycol solution (mw=8000) at final concentrations of 5- and25% v/v. In all cases, the Δ9 protein precipitated at either 80%ammonium sulfate or 25% polyethylene glycol. The resulting pellets werethen dialyzed extensively in 25 mM sodium phosphate buffer (pH 6.0).

Cation Exchange Chromotography: The solubilized pellet materialdescribed above was clarified via centrifugation and applied to Mono SHR10/10 column equilibrated in 25 mM sodium phosphate buffer (pH 6.0).After extensive column washing, basic proteins bound to the columnmatrix were eluted using a 0-500 mM NaCl gradient over 1 hr (2 ml/min: 2ml fractions). Typically, the Δ9 protein eluted between 260- and 350 mMNaCl., as determined by enzymatic and western analysis. After dialysis,this material was further fracionated by acyl carrier protein(ACP)-sepharose and phenyl superose chromatography.

Acyl Carrier Protein-Sepharose Chromatography: ACP was purchased fromSigma Chemical Company and purified via precipitation at pH 4.1 (Rockand Cronan, 1981 J. Biol. Chem. 254, 7116-7122) before linkage to thebeads. ACP-sepharose was prepared by covalently binding 100 mg of ACP tocyanogen bromide activated sepharose 4B beads, essentially as describedby Pharmacia, Inc., in the package insert. After linkage and blocking ofthe remaining sites with glycine, the ACP-sepharose material was packedinto a HR 5/5 column (Pharmacia, Inc.) and equilibrated in 25 mM sodiumphosphate buffer (pH 7.0). The dialyzed fractions identified above werethen loaded onto the column (McKeon and Stumpf, 1982 J. Biol. Chem. 257,12141-12147; Thompson et al., 1991, Proc. Natl. Acad. Sci. USA 88,2578-2582). After extensive column washing, ACP-binding proteins wereeluted using 1 M NaCl. Enzymatic and western analysis, followed by aminoterminal sequencing, indicated that the eluent contained Δ-9 protein.The Δ-9 protein purified from corn was determined to have a molecularsize of approximately 38 kDa by SDS-PAGE analysis (Hames, 1981 in GelElectrophoresis of Proteins: A Practical Approach, eds Hames B D andRickwood, D., IRL Press, Oxford).

Phenyl Sepharose Chromatography: The fractions containing Δ9 obtainedfrom the ACP-Sepharose column were adjusted to 0.4 M ammonium sulfate(25 mM sodium phosphate, pH 7.0) and loaded onto a Pharmacia PhenylSuperose column (HR 10/10). Proteins were eluted by running a gradient(0.4-0.0 M ammonium sulfate) at 2 ml/min for 1 hour. The Δ9 proteintypically eluted between 60- and 30 mM ammonium sulfate as determined byenzymatic and western analysis.

Example 32 Evidence for the Increase in Stearic Acid in Leaves as aResult of Transformation of Plants with Δ9 Desaturase Ribozymes

Part A Method used to determine the stearic acid levels in planttissues. The procedure for extraction and esterification of fatty acidsfrom plant tissue was modified from a described procedure (Browse et.al., 1986, Anal. Biochem. 152, 141-145). One to 20 mg of plant tissuewas placed in Pyrex 13 mm screw top test tubes. After addition of 1 mlof methanolic HCL (Supelco, Bellefonte, Pa.), the tubes were purged withnitrogen gas and sealed. The tubes were heated at 80° C. for 1 hour andallowed to cool. The heating in the presence of the methanolic HCLresults in the extraction as well as the esterification of the fattyacids. The fatty acid methyl esters were removed from the reactionmixture by extraction with hexane. One ml of hexane and 1 ml of 0.9%(w/v) NaCl was added followed by vigorous shaking of the test tubes.After centrifugation of the tubes at 2000 rpm for 5 minutes the tophexane layer was removed and used for fatty acid methyl ester analysis.Gas chromatograph analysis was performed by injection of 1 μl of thesample on a Hewlett Packard (Wilmington, Del.) Series II model 5890 gaschromatograph equipped with a flame ionization detector and a J&WScientific (Folsom, Calif.) DB-23 column. The oven temperature was 150°C. throughout the run and the flow of the carrier gas (helium) was 80cm/sec. The run time was 20 minutes. The conditions allowed for theseparation of the 5 fatty acid methyl esters of interest: C16:0,palmityl methyl ester; C18:0, stearyl methyl ester; C18:1, oleoyl methylester; C18:2, linolcoyl methyl ester; and C18:3, linolenyl methyl ester.Data collection and analysis was performed with a Hewlett Packard SeriesII Model 3396 integrator and a PE Nelson (Perkin Elmer, Norwalk, Conn.)data collection system. The percentage of each fatty acid in the samplewas taken directly from the readouts of the data collection system.Quantitative amounts of each fatty acid were calculated using the peakareas of a standard (Matreya, Pleasant Gap, Pa.) which consisted of aknown amount of the five fatty acid methyl esters. The amount calculatedwas used to estimate the percentage, of total fresh weight, representedby the five fatty acids in the sample. An adjustment was not made forloss of fatty acids during the extraction and esterification procedure.Recovery of the standard sample, after subjecting it to the extractionand esterification procedure (with no tissue present), ranged from 90 to100% depending on the original amount of the sample. The presence ofplant tissue in the extraction mixture had no effect on the recovery ofthe known amount of standard.

Part B Demonstration of an increase in stearic acid in leaves due tointroduction of Δ9 desaturase ribozymes. Leaf tissue from individualplants was assayed for stearic acid as described in Part A. A total of428 plants were assayed from 35 lines transfonned with active Δ9desaturase ribozymes (RPA85, RPA114, RPA118) and 406 plants from 31lines transformed with Δ9 desaturase inactive ribozymes (RPA113, RPA115,RPA119). Table XI summarizes the results obtained for stearic acidlevels in these plants. Seven percent of the plants from the activelines had stearic acid levels greater than 3%, and 2% had levels greaterthan 5%. Only 3% of the plants from the inactive lines had stearic acidlevels greater than 3%. Two percent of the control plants had leaveswith stearate greater than 3%. The controls included 49 non-transformedplants and 73 plants transformed with a gene not related to Δ9desaturase. There were no plants from the inactive lines or controlsthat had leaf stearate greater than 4%. Two of the lines transformedwith the active Δ9 desaturase ribozyme RPA85 produced many plants whichexhibited increased stearate in their leaves. Line RPA85-06 had 6 out ofthe 15 plants assayed with stearic acid levels which were between 3 and4%, about 2-fold greater than the average of the controls (FIG. 30) Theaverage stearic acid content of the control plants (122 plants) as 1.69%(SD+/−0.49%). The average stearic acid content of leaves from lineRPA85-06 as 2.86% (+/−0.57%). Line RPA85-15 had 6 out of 15 plantsassayed with stearic acid levels which were approximately 4-fold greaterthan the average of the controls (FIG. 31). The average leaf stearicacid content of line RPA85-15 was 3.83% (+/−2.53%). When the leafanalysis was repeated for RPA85-15 plants, the stearic acid level inleaves from plants previously shown to have normal stearic acid levelsremained normal and leaves from plants with high stearic acid were againfound to be high (FIG. 31). The stearic acid levels in leaves of plantsfrom two lines which were transformed with an inactive Δ9 desaturaseribozyme, RPA113, is shown in FIGS. 32 and 33. RPA113-06 had threeplants with a stearic acid content of 3% or higher. The average stcaricacid content of leaves from line RPA113-06 was 2.26% (+/−0.65%).RPA113-17 had no plants with leaf stearic acid content greater than 3%.The average stearic acid content of leaves from line RPA113-17 was 1.76%(+/−0.29%). The stearic acid content of leaves from 15 control plants isshown in FIG. 34. The average stearic acid content for these 15 controlplants was 1.70% (+/−0.6%). When compared to the control and inactive Δ9desaturase ribozyme data, the results obtained for stearic acid contentin RPA85-06 and RPA85-15 demonstrate an increase in stearic acid contentdue to the introduction of the Δ9 desaturase ribozyme.

Example 33 Inheritance of the High Stearic Acid Trait in Leaves

Part A Results obtained with stearic acid levels in leaves fromoffspring of high stearic acid plants. Plants from line RPA85-15 werepollinated as described herein. Twenty days after pollination zygoticembryos were excised from immature kernels from these RPA85-15 plantsand placed in a tube on media as described herein for growth ofregenerated plantlets. After the plants were transferred to thegreenhouse, fatty acid analysis was performed on the leaf tissue. FIG.35 shows the stearic acid levels of leaves from 10 different plants forone of the crosses, RPA85-15.07 selfed. Fifty percent of the plants hadhigh leaf stearic acid and 50% had normal leaf stearic acid. Table XIIshows the results from 5 different crosses of RPA85-15 plants. Thenumber of plants with high stearic acid ranged from 20 to 50%.

Part B Results demonstrating reductions in Δ9 desaturase levels in nextgeneration (R1) maize leaves expressing ribozymes targeted to Δ9desaturase mRNA. In next generation maize plants that showed a highstearate content (see above Part A), Δ9 desaturase was partiallypurified from R1 maize leaves, using the protocol described herein.Western analysis was performed on several of the high stearate plants.In leaves of next generation plants, a 40-50% reduction of Δ9 desaturasewas observed in those plants that had high stearate content (FIG. 36).The reduction was comparable to R0 maize leaves. This reduction wasobserved in either OQ414 plants crossed with RPA85-15 pollcn or RPA85-15plants crossed with self or siblings. Therefore, this suggests that thegene encoding the ribozyme is heritable.

Example 34 Increase in Stearic Acid in Plant Tissues Using Antisense-Δ9Desaturase

Part A Method for culturing somatic embryos of maize. The production andregeneration of maize embryogenic callus has been described herein.Somatic embryos make up a large part of this embryogenic callus. Thesomatic embryos continued to form in callus because the callus wastransferred every two weeks. The somatic embryos in embryogenic calluscontinued to proliferate but usually remained in an early stage ofembryo development because of the 2,4-D in the culture medium. Thesomatic embryos regenerated into plantlets because the callus wassubjected to a regeneration procedure described herein. Duringregeneration the somatic embryo formed a root and a shoot, and ceasesdevelopment as an embryo. Somatic embryos were made to develop as seedembryos, i.e., beyond the early stage of development found inembryogenic callus and no regeneration, by a specific medium treatment.This medium treatment involved transfer of the embryogenic callus to aMurashige and Skoog medium (MS; described by Murashige and Skoog in1962) which contains 6% (w/v) sucrose and no plant hormones. The calluswas grown on the MS medium with 6% sucrose for 7 days and then thesomatic embryos were individually transferred to MS medium with 6%sucrose and 10 μM abscisic acid (ABA). The somatic embryos were assayedfor fatty acid composition as described herein after 3 to 7 days ofgrowth on the ABA medium. The fatty acid composition of somatic embryosgrown on the above media was compared to the fatty acid composition ofembryogenic callus and maize zygotic embryos 12 days after pollination(Table XIII). The fatty acid composition of the somatic embryos wasdifferent than that of the embryogenic callus. The embryogenic callushad a higher percentage of C16:0 and C18:3, and a lower percentage ofC18:1 and C18:2. The percentage of lipid represented by the fresh weightwas different for the embryogenic callus when compared to the somaticembryos; 0.4% versus 4.0%. The fatty acid composition of the zygoticembryos and somatic embryos were very similar and their percentage oflipid represented by the fresh weight were nearly identical. It wasconcluded that the somatic embryo culture system described above wouldbe an useful in vitro system for testing the effect of certain genes onlipid synthesis in developing embryos of maize.

Part B Increase in stearic acid in somatic embryos of maize as a resultof the introduction of an antisense-Δ9 desaturase gene. Somatic embryoswere produced using the method described herein from embryogenic callustransformed with pDAB308/pDAB430. The somatic embryos from 16 differentlines were assayed for fatty acid composition. Two lines, 308/430-12 and308/430-15, were found to produce somatic embryos with high levels ofstearic acid. The stearic acid content of somatic embryos from these twolines is compared to the stearic acid content of somatic embryos fromtheir control lines in FIGS. 37 and 38. The control lines were from thesame culture that the transformed lines came from except that they werenot transformed. For line 308/430-12, stearic acid in somatic embryosranged from 1 to 23% while the controls ranged from 0.5 to 3%. For line308/430-15, stearic acid in somatic embryos ranged from 2 to 15% whilethe controls ranged from 0.5 to 3%. More than 50% of the somatic embryoshad stearic acid levels which were above the range of the controls inboth the transformed lines. The above results indicate that anantisense-Δ9 desaturase gene can be used to raise the stearic acidlevels in somatic embryos of maize.

Part C Demonstration of an increase in stearic acid in leaves due tointroduction of an antisense-Δ9 desaturase gene. Embryogenic culturesfrom lines 308/430-12 and 308/430-15 were used to regenerate plants.Leaves from these plants were analyzed for fatty acid composition usingthe method previously described. Only 4 plants were obtained from the308/430-15 culture and the stearic acid level in the leaves of theseplants were normal, 1-2%. The stearic acid levels in leaves from plantsof line 308/430-12 are shown in FIG. 39. The stearic acid levels inleaves ranged from 1 to 13% in plants from line 308/430-12. About 30% ofthe plants from line 308/430-12 had stearic acid levels above the rangeobserved in the controls, 1-2%. These results indicate that the stearicacid levels can be raised in leaves of maize by introduction of anantisense-Δ9 desaturase gene.

By “antisense” is meant a non-enzymatic nucleic acid molecule that bindsto a RNA (target RNA) by means of RNA-RNA or RNA-DNA or RNA-PNA (proteinnucleic acid; Egholm et al., 1993 Nature 365, 566) interactions andalters the activity of the target RNA (for a review see Stein and Cheng,1993 Science 261, 1004).

Example 35 Amylose Content Assay of Maize Pooled Starch Sample andSingle Kernel

The amylose content was assayed by the method of Hovenkamp-Hermelink etal. (Potato Research 31:241-246) with modifications. For pooled starchsample, 10 mg to 100 mg starch was dissolved in 5 ml 45% perchloric acidin plastic culture tube. The solution was mixed occasionally byvortexing. After one hour, 0.2 ml of the starch solution was diluted to10 ml by H₂O. 0.4 ml of the diluted solution was then mixed with 0.5 mldiluted Lugol's solution (Sigma) in 1 ml cuvet. Readings at 618 nm and550 nm were immediately taken and the R ratio (618 nm/550 nm) wascalculated. Using standard equation P (percentage ofamylose)=(4.5R-2.6)/(7.3-3R) generated from potato amylose and maizeamylopectin (Sigma, St. Louis), ainylose content was determined. Forfrozen single kernel sample, same procedure as above was used except itwas extracted in 45% perchloric acid for 20 min instead for one hour.

Example 36 Starch Purification and Granular Bound Starch Synthase (GBSS)Assay

The purification of starch and following GBSS activity assay weremodified from the methods of Shure et al. (Cell, 35:225-233, 1983) andNelson et al. (Plant Physiology, 62:383-386, 1978). Maize kernel washomogenized in 2 volume (v/w) of 50 mM Tris-HCl, pH 8.0, 10 mM EDTA andfiltrated through 120 μm nylon membrane. The material was thencentrifuged at 5000 g for 2 min and the supernatant was discarded. Thepellet was washed three times by resuspending in water and removingsupernatant by centrifugation. After washing, the starch was filtratedthrough 20 μm nylon membrane and centrifuged. Pellet was thenlyophilized and stored in −20° C. until used for activity assay.

A standard GBSS reaction mixture contained 0.2 M Tricine, pH 8.5, 25 mMGlutathione, 5 mM EDTA, 1 mM ¹⁴C ADPG (6 nci/μmol), and 10 mg starch ina total volume of 200 μl. Reactions were conducted at 37° C. for 5 minand terminated by adding 200 μl of 70% ethanol (v/v) in 0.1 M KCl. Thematerial was centrifuged and unincorporated ADPG in the supernatant isremoved. The pellet was then washed four time with 1 ml water each inthe same fashion. After washing, pellet was suspended in 500 μl water,placed into scintillation vial, and the incorporated ADPG was counted bya Beckman (Fullerton, Calif.) scintillation counter. Specific activitywas given as pmoles of ADPG incorporated into starch per min per mgstarch.

Example 37 Analysis of Antisense-GBSS Plants

Because of the segregation of R2 seeds, single kernels should thereforebe analyzed for amylose content to identify phenotype. Because of thelarge amount of samples generated in this study, a two-step screeningstrategy was used. In the first step, 30 kernels were taken randomlyfrom the same ear, freeze-dried and homogenized into starch flour.Amylose assays on the starch flours were carried out. Lines with reducedamylose content were identified by statistical analysis. In the secondstep, amylose content of the single kernels in the lines with reducedamylose content was further analyzed (25 to 50 kernels per ear). Twosets of controls were used in the screening, one of the sets wereuntransformed lines with the same genetic background and the other weretransformed lines which did not carry transgene due to segregation(Southern analysis negative line).

81 lines representing 16 transformation events were examined at thepooled starch level. Among those lines, six with significant reductionof amylose content by statistical analysis were identified for furthersingle kernel analysis. One line, 308/425-12.2.1, showed significantreduction of amylose content (FIG. 40).

Twenty five individual kernels of CQ806, a conventional maize inbredline, were analyzed. The amylose content of CQ806 ranged from 24.4% to32.2%, averaging 29.1%. The single kernel distribution of amylosecontent is skewed slightly towards lower amylose contents. Forty ninesingle kernels of 308/425-12.2.1.1 were analyzed. Given that308/425-12.2.1.1 resulted from self pollination of a hemizygousindividual, the expected distribution would consist of 4 distinctgenetic classes present in equal frequencies since endosperm is atriploid tissue. The 4 genetic classes consist of individuals carrying0, 1, 2, and 3 copies of the antisense construct. If there is a largedosage effect for the transgene, then the distribution of amylosecontents would be tetramodal. One of the modes of the resultingdistribution should be indistinguishable from the non-transgenic parent.If there is no dosage effect for the transgene (individuals carrying 1,2 or 3 copies of the transgene are phenotypically equivalent), then thedistribution should be bimodal with one of the modes identical to theparent. The number of individuals included in the modes should be 3:1 oftransgenic:parental. The distribution for 308/425-12.2.1.1 is distinctlytrimodal. The central mode is approximately twice the size of eitherother mode. The two distal modes are of approximately equal size.Goodness of fit to a 1:2:1 ratio was tested and the fit was excellent.

Further evidence was available demonstrating that the mode with thehighest amylose content was identical to the non-transgenic parent. Thiswas done using discriminant analysis. The CQ806 and 308/425-12.2.1.1data sets were combined for this analysis. The distance metrics used inthe analysis were calculated using amylose contents only. The estimatesof variance from the individual analyses were used in all tests. Nopooled estimate of variance was employed. The original data was testedfor reclassification. Based on the discriminant analysis, the entiremode of the 308/425-12.2.1.1 distribution with the highest amylosecontent would be more appropriately classified as parental. This isstrong confirmation that this mode of thc distribution is parental. Ofthe remaining two modes, the central mode is approximately twice thesize of the lowest amylose content mode. This would be expected if thecentral mode includes two genetic classes: individuals with 1 or 2copies of the antisense construct. The mode with the lowest amylosecontent thus represents those individuals which are fully homozygous (3copies) for the antisense construct. The 2:1 ratio was tested and couldnot be rejected on the basis of the data.

This analysis indicates that the antisense GBSS gene as functioning in308/425-12.2.1.1 demonstrates a dosage dependent reduction in amylosecontent of maize kernels.

Example 38 Analysis of Ribozyme-GBSS Plants

The same two-step screening strategy as in the antisense study (Example37) was used to analyze ribozyme-GBSS plants. 160 lines representing 11transformation events were examined in the pooled starch level. Amongthe control lines (both untransformed line and Southern negative line),the amylose content varied from 28% to 19%. No significant reduction wasobserved among all lines carrying ribozyme gene (Southern positiveline). More than 20 selected lines were further analyzed in the singlekernel level, no significant amylose reduction as well as segregationpattern were found. It was apparent that ribozyme did not cause anyalternation in the phenotypic level.

Transformed lines were further examined by their GBSS activity (asdescribed in Example 36). For each line, 30 kernels were taken from thefrozen ear and starch was purified. Table XIV shows the results of 9plants representing one transformation event of the GBSS activity in thepooled starch samples, amylose content in the pooled starch samples, andSouthern analysis results. Three southern negative lines: RPA63.0283,RPA63.0236, and RPA63.0219 were used as control.

The GBSS activities of control lines RPA63.0283, RPA63.0236, andRPA63.0219 were around 300 units/mg starch, In lines RPA63.021 1,RPA63.021 8, RPA63.0209, and RPA63.0210, a reduction of GBSS activity tomore than 30% was observed. The correlation of varied GBSS activity tothe Southern analysis in this group (from RPA63.0314 to RPA63.0210 ofTable XIV) indicated that the reduced GBSS activity was caused by theexpression of ribozyme gene incorporated into the maize genome.

GBSS activities at the single kernel level of line RPA63.0218 (Southernpositive and reduced GBSS activity in pooled starch) was furtherexamined, using RPA63.0306 (Southern negative and GBSS activity normalin pooled starch) as control. About 30 kernels from each line weretaken, and starch samples were purified from each kernel individually.FIG. 41 clearly indicated reduced GBSS activity in line RPA63.0218compared to RPA63.0306.

Other embodiments are within the following claims.

Characteristics of naturally occurring ribozymes Group I Introns Size:˜150 to >1000 nucleotides. Requires a U in the target sequenceimmediately 5′ of the cleavage site. Binds 4-6 nucleotides at the5′-side of the cleavage site. Reaction mechanism: attack by the 3′-OH ofguanosine to generate cleavage products with 3′-OH and 5′-guanosine.Additional protein cofactors required in some cases to help folding andmaintainance of the active structure [1]. Over 300 known members of thisclass. Found as an intervening sequence in Tetrahymena thermophila rRNA,fungal mitochondria, chloroplasts, phage T4, blue-green algae, andothers. Major structural features largely established throughphylogenetic comparisons, mutagenesis, and biochemical studies [2, 3].Complete kinetic framework established for one ribozyme [4, 5, 6, 7].Studies of ribozyme folding and substrate docking underway [8, 9, 10].Chemical modification investigation of important residues wellestablished [11, 12]. The small (4-6 nt) binding site may make thisribozyme too non-specific for targeted RNA cleavage, however, theTetrahymena group I intron has been used to repair a “defective”β-galactosidase message by the ligation of new β-galactosidase sequencesonto the defective message [13]. RNAse P RNA (M1 RNA) Size: ˜290 to 400nucleotides. RNA portion of a ubiquitous ribonucleoprotein enzyme.Cleaves tRNA precursors to form mature tRNA [14]. Reaction mechanism:possible attack by M²⁺-OH to generate cleavage products with 3′-OH and5′-phosphate. RNAse P is found throughout the prokaryotes andeukaryotes. The RNA subunit has been sequenced from bacteria, yeast,rodents, and primates. Recruitment of endogenous RNAse P for therapeuticapplications is possible through hybridization of an External GuideSequence (EGS) to the target RNA [15, 16] Important phosphate and 2′ OHcontacts recently identified [17, 18] Group II Introns Size: >1000nucleotides. Trans cleavage of target RNAs recently demonstrated [19,20]. Sequence requirements not fully determined. Reaction mechanism:2′-OH of an internal adenosine generates cleavage products with 3′-OHand a “lariat” RNA containing a 3′-5′ and a 2′-5′ branch point. Onlynatural ribozyme with demonstrated participation in DNA cleavage [21,22] in addition to RNA cleavage and ligation. Major structural featureslargely established through phylogenetic comparisons [23]. Important 2′OH contacts beginning to be identified [24] Kinetic framework underdevelopment [25] Neurospora VS RNA Size: ˜144 nucleotides. Transcleavage of hairpin target RNAs recently demonstrated [26]. Sequencerequirements not fully determined. Reaction mechanism: attack by 2′-OH5′ to the scissile bond to generate cleavage products with 2′,3′-cyclicphosphate and 5′-OH ends. Binding sites and structural requirements notfully determined. Only 1 known member of this class. Found in NeurosporaVS RNA. Hammerhead Ribozyme (see text for references) Size: ˜13 to 40nucleotides. Requires the target sequence UH immediately 5′ of thecleavage site. Binds a variable number nucleotides on both sides of thecleavage site. Reaction mechanism: attack by 2′-OH 5′ to the scissilebond to generate cleavage products with 2′,3′-cyclic phosphate and 5′-OHends. 14 known members of this class. Found in a number of plantpathogens (virusoids) that use RNA as the infectious agent. Essentialstructural features largely defined, including 2 crystal structures [ ]Minimal ligation activity demonstrated (for engineering through in vitroselection) [ ] Complete kinetic framework established for two or moreribozymes [ ]. Chemical modification investigation of important residueswell established [ ]. Hairpin Ribozyme Size: ˜50 nucleotides. Requiresthe target sequence GUC immediately 3′ of the cleavage site. Binds 4-6nucleotides at the 5′-side of the cleavage site and a variable number tothe 3′-side of the cleavage site. Reaction mechanism: attack by 2′-OH 5′to the scissile bond to generate cleavage products with 2′,3′-cyclicphosphate and 5′-OH ends. 3 known members of this class. Found in threeplant pathogen (satellite RNAs of the tobacco ringspot virus, arabismosaic virus and chicory yellow mottle virus) which uses RNA as theinfectious agent. Essential structural features largely defined [27, 28,29, 30] Ligation activity (in addition to cleavage activity) makesribozyme amenable to engineering through in vitro selection [31]Complete kinetic framework established for one ribozyme [32]. Chemicalmodification investigation of important residues begun [33, 34].Hepatitis Delta Virus (HDV) Ribozyme Size: ˜60 nucleotides. Transcleavage of target RNAs demonstrated [35]. Binding sites and structuralrequirements not fully determined, although no sequences 5′ of cleavagesite are required. Folded ribozyme contains a pseudoknot structure [36].Reaction mechanism: attack by 2′-OH 5′ to the scissile bond to generatecleavage products with 2′,3′-cyclic phosphate and 5′-OH ends. Only 2known members of this class. Found in human HDV. Circular form of HDV isactive and shows increased nuclease stability [37] 1. Mohr, G.; Caprara,M. G.; Guo, Q.; Lambowitz, A. M. Nature, 370, 147-150 (1994). 2. Michel,Francois; Westhof, Eric. Slippery substrates. Nat. Struct. Biol. (1994),1(1), 5-7. 3. Lisacek, Frederique; Diaz, Yolande; Michel, Francois.Automatic identification of group I intron cores in genomic DNAsequences. J. Mol. Biol. (1994), 235(4), 1206-17. 4. Herschlag, Daniel;Cech, Thomas R.. Catalysis of RNA cleavage by the Tetrahymenathermophila ribozyme. 1. Kinetic description of the reaction of an RNAsubstrate complementary to the active site. Biochemistry (1990), 29(44),10159-71. 5. Herschlag, Daniel; Cech, Thomas R.. Catalysis of RNAcleavage by the Tetrahymena thermophila ribozyme. 2. Kinetic descriptionof the reaction of an RNA substrate that forms a mismatch at the activesite. Biochemistry (1990), 29(44), 10172-80. 6. Knitt, Deborah S.;Herschlag, Daniel. pH Dependencies of the Tetrahymena Ribozyme Reveal anUnconventional Origin of an Apparent pKa. Biochemistry (1996), 35(5),1560-70. 7. Bevilacqua, Philip C.; Sugimoto, Naoki; Turner, Douglas H..A mechanistic framework for the second step of splicing catalyzed by theTetrahymena ribozyme. Biochemistry (1996), 35(2), 648-58. 8. Li, Yi;Bevilacqua, Philip C.; Mathews, David; Turner, Douglas H.. Thermodynamicand activation parameters for binding of a pyrene-labeled substrate bythe Tetrahymena ribozyme: docking is not diffusion-controlled and isdriven by a favorable entropy change. Biochemistry (1995), 34(44),14394-9. 9. Banerjee, Aloke Raj; Turner, Douglas H.. The time dependenceof chemical modification reveals slow steps in the folding of a group Iribozyme. Biochemistry (1995), 34(19), 6504-12. 10. Zarrinkar, PatrickP.; Williamson, James R.. The P9.1-P9.2 peripheral extension helps guidefolding of the Tetrahymena ribozyme. Nucleic Acids Res. (1996), 24(5),854-8. 11. Strobel, Scott A.; Cech, Thomas R.. Minor groove recognitionof the conserved G.cntdot.U pair at the Tetrahymena ribozyme reactionsite. Science (Washington, D.C.) (1995), 267(5198), 675-9. 12. Strobel,Scott A.; Cech, Thomas R.. Exocyclic Amine of the Conserved G.cntdot.UPair at the Cleavage Site of the Tetrahymena Ribozyme Contributes to5′-Splice Site Selection and Transition State Stabilization.Biochemistry (1996), 35(4), 1201-11. 13. Sullenger, Bruce A.; Cech,Thomas R.. Ribozyme-mediated repair of defective mRNA by targetedtrans-splicing. Nature (London) (1994), 371(6498), 619-22. 14.Robertson, H. D.; Altman, S.; Smith, J. D. J. Biol. Chem., 247,5243-5251 (1972). 15. Forster, Anthony C.; Altman, Sidney. Externalguide sequences for an RNA enzyme. Science (Washington, D.C., 1883-)(1990), 249(4970), 783-6. 16. Yuan, Y.; Hwang, E. S.; Altman, S.Targeted cleavage of mRNA by human RNase P. Proc. Natl. Acad. Sci. USA(1992) 89, 8006-10. 17. Harris, Michael E.; Pace, Norman R..Identification of phosphates involved in catalysis by the ribozyme RNaseP RNA. RNA (1995), 1(2), 210-18. 18. Pan, Tao; Loria, Andrew; Zhong,Kun. Probing of tertiary interactions in RNA: 2′-hydroxyl-base contactsbetween the RNase P RNA and pre-tRNA. Proc. Natl. Acad. Sci. U.S.A.(1995), 92(26), 12510-14. 19. Pyle, Anna Marie; Green, Justin B..Building a Kinetic Framework for Group II Intron Ribozyme Activity:Quantitation of Interdomain Binding and Reaction Rate. Biochemistry(1994), 33(9), 2716-25. 20. Michels, William J. Jr.; Pyle, Anna Marie.Conversion of a Group II Intron into a New Multiple-Turnover Ribozymethat Selectively Cleaves Oligonucleotides: Elucidation of ReactionMechanism and Structure/Function Relationships. Biochemistry (1995),34(9), 2965-77. 21. Zimmerly, Steven; Guo, Huatao; Eskes, Robert; Yang,Jian; Perlman, Philip S.; Lambowitz, Alan M.. A group II intron RNA is acatalytic component of a DNA endonuclease involved in intron mobility.Cell (Cambridge, Mass.) (1995), 83(4), 529-38. 22. Griffin, Edmund A.,Jr.; Qin, Zhifeng; Michels, Williams J., Jr.; Pyle, Anna Marie. Group IIintron ribozymes that cleave DNA and RNA linkages with similarefficiency, and lack contacts with substrate 2′-hydroxyl groups. Chem.Biol. (1995), 2(11), 761-70. 23. Michel, Francois; Ferat, Jean Luc.Structure and activities of group II introns. Annu. Rev. Biochem.(1995), 64, 435-61. 24. Abramovitz, Dana L.; Friedman, Richard A.; Pyle,Anna Marie. Catalytic role of 2′-hydroxyl groups within a group IIintron active site. Science (Washington, D.C.) (1996), 271(5254),1410-13. 25. Daniels, Danette L.; Michels, William J., Jr.; Pyle, AnnaMarie. Two competing pathways for self-splicing by group II introns: aquantitative analysis of in vitro reaction rates and products. J. Mol.Biol. (1996), 256(1), 31-49. 26. Guo, Hans C. T.; Collins, Richard A..Efficient trans-cleavage of a stem-loop RNA substrate by a ribozymederived from Neurospora VS RNA. EMBO J. (1995), 14(2), 368-76. 27.Hampel, Arnold; Tritz, Richard; Hicks, Margaret; Cruz, Phillip.‘Hairpin’ catalytic RNA model: evidence for helixes and sequencerequirement for substrate RNA. Nucleic Acids Res. (1990), 18(2),299-304. 28. Chowrira, Bharat M.; Berzal-Herranz, Alfredo; Burke, JohnM.. Novel guanosine requirement for catalysis by the hairpin ribozyme.Nature (London) (1991), 354(6351), 320-2. 29. Berzal-Herranz, Alfredo;Joseph, Simpson; Chowrira, Bharat M.; Butcher, Samuel E.; Burke, JohnM.. Essential nucleotide sequences and secondary structure elements ofthe hairpin ribozyme. EMBO J. (1993), 12(6), 2567-73. 30. Joseph,Simpson; Berzal-Herranz, Alfredo; Chowrira, Bharat M.; Butcher, SamuelE.. Substrate selection rules for the hairpin ribozyme determined by invitro selection, mutation, and analysis of mismatched substrates. GenesDev. (1993), 7(1), 130-8. 31. Berzal-Herranz, Alfredo; Joseph, Simpson;Burke, John M.. In vitro selection of active hairpin ribozymes bysequential RNA-catalyzed cleavage and ligation reactions. Genes Dev.(1992), 6(1), 129-34. 32. Hegg, Lisa A.; Fedor, Martha J.. Kinetics andThermodynamics of Intermolecular Catalysis by Hairpin Ribozymes.Biochemistry (1995), 34(48), 15813-28. 33. Grasby, Jane A.; Mersmann,Karin; Singh, Mohinder; Gait, Michael J.. Purine Functional Groups inEssential Residues of the Hairpin Ribozyme Required for CatalyticCleavage of RNA. Biochemistry (1995), 34(12), 4068-76. 34. Schmidt,Sabine; Beigelman, Leonid; Karpeisky, Alexander; Usman, Nassim;Sorensen, Ulrik S.; Gait, Michael J.. Base and sugar requirements forRNA cleavage of essential nucleoside residues in internal loop B of thehairpin ribozyme: implications for secondary structure. Nucleic AcidsRes. (1996), 24(4), 573-81. 35. Perrotta, Anne T.; Been, Michael D..Cleavage of oligoribonucleotides by a ribozyme derived from thehepatitis .delta. virus RNA sequence. Biochemistry (1992), 31(1), 16-21.36. Perrotta, Anne T.; Been, Michael D.. A pseudoknot-like structurerequired for efficient self-cleavage of hepatitis delta virus RNA.Nature (London) (1991), 350(6317), 434-6. 37. Puttaraju, M.; Perrotta,Anne T.; Been, Michael D.. A circular trans-acting hepatitis delta virusribozyme. Nucleic Acids Res. (1993), 21(18), 4253-8.

TABLE II 2.5 μmol RNA Synthesis Cycle Wait Reagent Equivalents AmountTime* Phosphoramidites 6.5 163 μL 2.5 S-Ethyl Tetrazole 23.8 238 μL 2.5Acetic Anhydride 100 233 μL  5 sec N-Methyl Imidazole 186 233 μL  5 secTCA 83.2 1.73 mL 21 sec Iodine 8.0 1.18 mL 45 sec Acetonitrile NA 6.67mL NA *Wait time does not include contact time during delivery.

TABLE IIIA GBSS Hammerhead Substrate Sequence nt. Position Substrate  12CGAUCGAUC GCCACAGC  68 GAAGGAAUA AACUCACU  73 AAUAAACUC ACUGCCAG  103AGAAGUGUA CUGCUCCG  109 GUACUGCUC CGUCCACC  113 UGCUCCGUC CACCAGUG  146GGGCUGCUC AUCUCGUC  149 CUGCUCAUC UCGUCGAC  151 GCUCAUCUC GUCGACGA  154CAUCUCGUC GACGACCA  169 CAGUGGAUU AAUCGGCA  170 AGUGGAUUA AUCGGCAU  173GGAUUAAUC GGCAUGGC  186 UGGCGGCUC UAGCCACG  188 GCGGCUCUA GCCACGUC  196AGCCACGUC GCAGCUCG  203 UCGCAGCUC GUCGCAAC  206 CAGCUCGUC GCAACGCG  230CUGGGCGUC CCGGACGC  241 GGACGCGUC CACGUUCC  247 GUCCACGUU CCGCCGCG  248UCCACGUUC CGCCGCGG  292 GACGGCGUC GGCGGCGG  308 GACACGCUC AGCAUUCG  314CUCAGCAUU CGGACCAG  315 UCAGCAUUC GGACCAGC  344 CCCAGGCUC CAGCACCA  385GGCCAGGUU CCCGUCGC  386 GCCAGGUUC CCGUCGCU  391 GUUCCCGUC GCUCGUCG  395CCGUCGCUC GUCGUGUG  398 UCGCUCGUC GUGUGCGC  425 AUGAACGUC GUCUUCGU  428AACGUCGUC UUCGUCGG  430 CGUCGUCUU CGUCGGCG  431 GUCGUCUUC GUCGGCGC  434GUCUUCGUC GGCGCCGA  473 GGCGGCCUC GGCGACGU  482 GGCGACGUC CUCGGCGG  485GACGUCCUC GGCGGCCU  527 CACCGUGUC AUGGUCGU  533 GUCAUGGUC GUCUCUCC  536AUGGUCGUC UCUCCCCG  538 GGUCGUCUC UCCCCGCU  540 UCGUCUCUC CCCGCUAC  547UCCCCGCUA CGACCAGU  556 CGACCAGUA CAAGGACG  581 ACCAGCGUC GUGUCCGA  586CGUCGUGUC CGAGAUCA  593 UCCGAGAUC AAGAUGGG  610 AGACAGGUA CGAGACGG  620GAGACGGUC AGGUUCUU  625 GGUCAGGUU CUUCCACU  626 GUCAGGUUC UUCCACUG  628CAGGUUCUU CCACUGCU  629 AGGUUCUUC CACUGCUA  637 CCACUGCUA CAAGCGCG  661CCGCGUGUU CGUUGACC  662 CGCGUGUUC GUUGACCA  665 GUGUUCGUU GACCACCC  679CCCACUGUU CCUGGAGA  680 CCACUGUUC CUGGAGAG  692 GAGAGGGUU UGGGGAAA  693AGAGGGUUU GGGGAAAG  716 GAGAAGAUC UACGGGCC  718 GAAGAUCUA CGGGCCUG  742AACGGACUA CAGGGACA  763 GCUGCGGUU CAGCCUGC  764 CUGCGGUUC AGCCUGCU  773AGCCUGCUA UGCCAGGC  788 GCAGCACUU GAAGCUCC  795 UUGAAGCUC CAAGGAUC  803CCAAGGAUC CUGAGCCU  812 CUGAGCCUC AACAACAA  826 CAACCCAUA CUUCUCCG  829CCCAUACUU CUCCGGAC  830 CCAUACUUC UCCGGACC  832 AUACUUCUC CGGACCAU  841CGGACCAUA CGGGGAGG  854 GAGGACGUC GUGUUCGU  859 CGUCGUGUU CGUCUGCA  860GUCGUGUUC GUCUGCAA  863 GUGUUCGUC UGCAACGA  888 CCGGCCCUC UCUCGUGC  890GGCCCUCUC UCGUGCUA  892 CCCUCUCUC GUGCUACC  898 CUCGUGCUA CCUCAAGA  902UGCUACCUC AAGAGCAA  913 GAGCAACUA CCAGUCCC  919 CUACCAGUC CCACGGCA  929CACGGCAUC UACAGGGA  931 CGGCAUCUA CAGGGACG  951 AGACCGCUU UCUGCAUC  952GACCGCUUU CUGCAUCC  953 ACCGCUUUC UGCAUCCA  959 UUCUGCAUC CACAACAU  968CACAACAUC UCCUACCA  970 CAACAUCUC CUACCAGG  973 CAUCUCCUA CCAGGGCC  985GGGCCGGUU CGCCUUCU  986 GGCCGGUUC GCCUUCUC  991 GUUCGCCUU CUCCGACU  992UUCGCCUUC UCCGACUA  994 CGCCUUCUC CGACUACC 1000 CUCCGACUA CCCGGAGC 1016CUGAACCUC CCGGAGAG 1027 GGAGAGAUU CAAGUCGU 1028 GAGAGAUUC AAGUCGUC 1033AUUCAAGUC GUCCUUCG 1036 CAAGUCGUC CUUCGAUU 1039 GUCGUCCUU CGAUUUCA 1040UCGUCCUUC GAUUUCAU 1044 CCUUCGAUU UCAUCGAC 1045 CUUCGAUUU CAUCGACG 1046UUCGAUUUC AUCGACGG 1049 GAUUUCAUC GACGGCUA 1057 CGACGGCUA CGAGAAGC 1085CGGAAGAUC AACUGGAU 1106 GCCGGGAUC CUCGAGGC 1109 GGGAUCCUC GAGGCCGA 1124GACAGGGUC CUCACCGU 1127 AGGGUCCUC ACCGUCAG 1133 CUCACCGUC AGCCCCUA 1141CAGCCCCUA CUACGCCG 1144 CCCCUACUA CGCCGAGG 1157 GAGGAGCUC AUCUCCGG 1160GAGCUCAUC UCCGGCAU 1162 GCUCAUCUC CGGCAUCG 1169 UCCGGCAUC GCCAGGGG 1187UGCGAGCUC GACAACAU 1196 GACAACAUC AUGCGCCU 1205 AUGCGCCUC ACCGGCAU 1214ACCGGCAUC ACCGGCAU 1223 ACCGGCAUC GUCAACGG 1226 GGCAUCGUC AACGGCAU 1241AUGGACGUC AGCGAGUG 1270 GGACAAGUA CAUCGCCG 1274 AAGUACAUC GCCGUGAA 1285CGUGAAGUA CGACGUGU 1294 CGACGUGUC GACGGCCG 1346 GCGGAGGUC GGGCUCCC 1352GUCGGGCUC CCGGUGGA 1370 CGGAACAUC CCGCUGGU 1384 GGUGGCGUU CAUCGGCA 1385GUGGCGUUC AUCGGCAG 1388 GCGUUCAUC GGCAGGCU 1421 CCCGACGUC AUGGCGGC 1436GCCGCCAUC CCGCAGCU 1445 CCGCAGCUC AUGGAGAU 1472 GUGCAGAUC GUUCUGCU 1475CAGAUCGUU CUGCUGGG 1476 AGAUCGUUC UGCUGGGC 1501 GAAGAAGUU CGAGCGCA 1502AAGAAGUUC GAGCGCAU 1514 CGCAUGCUC AUGAGCGC 1534 GGAGAAGUU CCCAGGCA 1535GAGAAGUUC CCAGGCAA 1559 GCCGUGGUC AAGUUCAA 1564 GGUCAAGUU CAACGCGG 1565GUCAAGUUC AACGCGGC 1589 CACCACAUC AUGGCCGG 1610 GACGUGCUC GCCGUCAC 1616CUCGCCGUC ACCAGCCG 1627 CAGCCGCUU CGAGCCCU 1628 AGCCGCUUC GAGCCCUG 1643UGCGGCCUC AUCCAGCU 1646 GGCCUCAUC CAGCUGCA 1666 GAUGCGAUA CGGAACGC 1690CUGCGCGUC CACCGGUG 1703 GGUGGACUC GUCGACAC 1706 GGACUCGUC GACACCAU 1715GACACCAUC AYCGAAGG 1718 ACCAYCAYC GAAGGCAA 1735 GACCGGGUU CCACAUGG 1736ACCGGGUUC CACAUGGG 1751 GGCCGCCUC AGCGUCGA 1757 CUCAGCGUC GACUGCAA 1769UGCAACGUC GUGGAGCC 1787 GCGGACGUC AAGAAGGU 1807 CACCACCUU GCAGCGCG 1820CGCGCCAUC AAGGUGGU 1829 AAGGUGGUC GGCACGCC 1843 GCCGGCGUA CGAGGAGA 1871UGCAUGAUC CAGGAUCU 1878 UCCAGGAUC UCUCCUGG 1880 CAGGAUCUC UCCUGGAA 1882GGAUCUCUC CUGGAAGG 1922 GUGCUGCUC AGCCUCGG 1928 CUCAGCCUC GGGGUCGC 1934CUCGGGGUC GCCGGCGG 1955 CCAGGGGUC GAAGGCGA 1970 GAGGAGAUC GCGCCGCU 1979GCGCCGCUC GCCAAGGA 2012 UGAAGAGUU CGGCCUGC 2013 GAAGAGUUC GGCCUGCA 2033CCCCUGAUC UCGCGCGU 2035 CCUGAUCUC GCGCGUGG 2055 AAACAUGUU GGGACAUC 2063UGGGACAUC UUCUUAUA 2065 GGACAUCUU CUUAUAUA 2066 GACAUCUUC UUAUAUAU 2068CAUCUUCUU AUAUAUGC 2069 AUCUUCUUA UAUAUGCU 2071 CUUCUUAUA UAUGCUGU 2073UCUUAUAUA UGCUGUUU 2080 UAUGCUGUU UCGUUUAU 2081 AUGCUGUUU CGUUUAUG 2082UGCUGUUUC GUUUAUGU 2085 UGUUUCGUU UAUGUGAU 2086 GUUUCGUUU AUGUGAUA 2087UUUCGUUUA UGUGAUAU 2094 UAUGUGAUA UGGAVAAG 2104 GGACAAGUA UGUGUAGC 2110GAUAGUGUA GCUGCUUG 2117 UAGCUGCUU GCUUGUGC 2121 UGCUUGCUU GUGCUAGU 2127CUUGUGCUA GUGUAAUA 2132 GCUAGUGUA AUAUAGUG 2135 AGUGUAAUA UAGUGUAG 2137UGUAAUAUA GUGUAGUG 2142 UAUAGUGUA GUGGUGGC 2165 CACAACCUA AUAAGCGC 2168AACCUAAUA AGCGCAUG 2181 CAUGAACUA AUUGCUUG 2184 GAACUAAUU GCUUGCGU 2188UAAUUGCUU GCGUGUGU 2197 GCGUGUGUS GUUAAGUA 2200 UGUGUAGUU AAGUACCG 2201GUGUAGUUA AGUACCGA 2205 AGUUAAGUA CCGAUCGG 2211 GUACCGAUC GGUAAUUU 2215CGAUCGGUA AUUUUAUA 2218 UCGGUAAUU UUAUAUUG 2219 CGGUAAUUU UAUAUUGC 2220GGUAAUUUU AUAUUGCG 2221 GUAAUUUUA UAUUGCGA 2223 AAUUUUAUA UUGCGAGU 2225UUUUAUAUU GCGAGUAA 2232 UUGCGAGUA AAUAAAUG 2236 GAGUAAAUA AAUGGACC 2248GGACCUGUA GUGGUGGA

TABLE III B Hammerhead Robozyme Sequence Targeted Against GBSS mRNA nt.Position HH Ribozyme Sequence  12 UGGCUGUGGC CUGAUGA X GAA AUCGAUCGGU 68 GCAGUGAGUU CUGAUGA X GAA AUUCCUUCCU  73 GGCUGGCAGU CUGAUGA X GAAAGUUUAUUCC  103 GACGGAGCAG CUGAUGA X GAA ACACUUCUCC  109 CUGGUGGACGCUGAUGA X GAA AGCAGUACAC  113 CGCACUGGUG CUGAUGA X GAA ACGGAGCAGU  146UCGACGAGAU CUGAUGA X GAA AGCAGCCCUG  149 UCGUCGACGA CUGAUGA X GAAAUGAGCAGCC  151 GGUCGUCGAC CUGAUGA X GAA AUGAGCAGCC  154 ACUGGUCGUCCUGAUGA X GAA ACGAGAUGAG  169 CAUGCCGAUU CUGAUGA X GAA AUCCACUGGU  170CCAUGCCGAU CUGAUGA X GAA AUCCACUGGU  173 CCGCCAUGCC CUGAUGA X GAAAUUAAUCCAC  186 GACGUGGCUA CUGAUGA X GAA AGCCGCCAUG  188 GCGACGUGGCCUGAUGA X GAA AGAGCCGCCA  196 GACGAGCUGC CUGAUGA X GAA ACGUGGCUAG  203GCGUUGCGAC CUGAUGA X GAA AGCUGCGACG  206 CGCGCGUUGC CUGAUGA X GAAACGAGCUGCG  230 ACGCGUCCGG CUGAUGA X GAA ACGCCCAGGC  241 GCGGAACGUGCUGAUGA X GAA ACGCGUCCGG  247 GCCGCGGCGG CUGAUGA X GAA ACGUGGACGC  248CGCCGCGGCG CUGAUGA X GAA AACGUGGACG  292 GUCCGCCGCC CUGAUGA X GAAACGCCGUCCG  308 UCCGAAUGCU CUGAUGA X GAA AGCGUGUCCG  314 CGCUGGUCCGCUGAUGA X GAA AUGCUGAGCG  315 GCGCUGGUCC CUGCUGA X GAA AAUGCUGAGC  344GCUGGUGCUG CUGAUGA X GAA AGCCUGGGCG  385 GAGCGACGGG CUGAUGA X GAAACCUGGCCCC  386 CGAGCGACGG CUGAUGA X GAA AACCUGGCCC  391 CACGACGAGCCUGAUGA X GAA ACGGGAACCU  395 CGCACACGAC CUGAUGA X GAA AGCGACGGGA  398UGGCGCACAC CUGAUGA X GAA ACGAGCGACG  425 CGACGAAGAC CUGAUGA X GAAACGUUCAUGC  428 CGCCGACGAA CUGAUGA X GAA AGACGACGUU  430 GGCGCCGACGCUGAUGA X GAA AGACGACGUU  431 CGGCGCCGAC CUGAUGA X GAA AAGACGACGU  434UCUCGGCGCC CUGAUGA X GAA ACGAAGACGA  473 GGACGUCGCC CUGAUGA X GAAAGGCCGCCGG  482 GGCCGCCGAG CUGAUGA X GAA ACGUCGCCGA  485 GCAGGCCGCCCUGAUGA X GAA AGGACGUCGC  527 AGACGACCAU CUGAUGA X GAA ACACGGUGCC  533GGGGAGAGAC CUGAUGA X GAA ACCAUGACAC  536 AGCGGGGAGA CUGAUGA X GAAACGACCAUGA  538 GUAGCGGGGA CUGAUGA X GAA AGACGACCAU  540 UCGUAGCGGGCUGAUGA X GAA AGAGACGACC  547 GUACUGGUCG CUGAUGA X GAA AGCGGGGAGA  556GGCGUCCUUG CUGCUGA X GAA ACUGGUCGUA  581 UCUCGGACAC CUGAUGA X GAAACGCUGGUGU  586 CUUGAUCUCG CUGAUGA X GAA ACACGACGCU  593 CUCCCAUCUUCUGAUGA X GAA AUCUCGGACA  610 GACCGUCUCG CUGAUGA X GAA ACCUGUCUCC  620GGAAGAACCU CUGAUGA X GAA ACCGUCUCGU  625 GCAGUGGAAG CUGAUGA X GAAACCUGACCGU  626 AGCAGUGGAA CUGAUGA X GAA AACCUGACCG  628 GUAGCACUGGCUGAUGA X GAA AGAACCUGAC  629 UGUAGCAGUG CUGAUGA X GAA AAGAACCUGA  637UCCGCGCUUG CUGAUGA X GAA AGCAGUGGAA  661 GUGGUCAACG CUGAUGA X GAAACACGCGGUC  662 GGUGGUCAAC CUGAUGA X GAA AACACGCGGU  665 GUGGGUGGUCCUGAUGA X GAA ACGAACACGC  679 CCUCUCCAGG CUGAUGA X GAA ACAGUGGGUG  680CCCUCUCCAH CUGAUGA X GAA AACAGUGGGU  692 UCUUUCCCCA CUGAUGA X GAAACCCUCUCCA  693 GUCUUUCCCC CUGAUGA X GAA AACCCUCUCC  716 CAGGCCCGUACUGAUGA X GAA AUCUUCUCCU  718 GUCAGGCCCG CUGAUGA X GAA AGAUCUUCUC  742GUUGUCCCUG CUGAUGA X GAA AGUCCGUUCC  763 UAGCAGGCUG CUGAUGA X GAAACCGCAGCUG  764 AUAGCAGGCU CUGAUGA X GAA AACCGCAGCU  773 CUGCCUGGCACUGAUGA X GAA AGCAGGCUGA  788 UUGGAGCUUC CUGAUGA X GAA AGUGCUGCCU  795AGGAUCCUUG CUGAUGA X GAA AGCUUCAAGU  803 UGAGGCUCAG CUGAUGA X GAAAUCCUUGGAG  812 GGUUGUUGUU CUGAUGA X GAA AGGCUCAGGA  826 UCCGGAGAAGVUGAUGA X GAA AUGGGUUGUU  829 UGGUCCGGAG CUGAUGA X GAA AGUAUGGGUU  830AUGGUCCGGA CUGAUGA X GAA AAGUAUGGGC  832 GUAUGGUCCG CUGAUGA X GAAAGAACUAUGG  841 GUCCUCCCCG CUGAUGA X GAA AUGGUCCGGA  854 AGACGAACACCUGAUGA X GAA ACGUCCUCCC  859 GUUGCAGACG CUGAUGA X GAA ACACGACGUC  860CGUUGCAGAC CUGAUGA X GAA AACACGACGU  863 AGUCGUUGCA CUGAUGA X GAAACGAACACGA  888 UAGCACGAGA CUGAUGA X GAA AGGGCCGGUG  890 GGUAGCACGACUGAUGA X GAA AGAGGGCCGG  892 GAGGUAGCAC CUGUAGA x GAA AGAGAGGGCC  898GCUCUUGAGG CUGAUGA X GAA AGCACGAGAG  902 AGUUGCUCUU CUGAUGA X GAAAGGUAGCACG  913 GUGGGACUGG CUGAUGA X GAA AGUUGCUCUU  919 GAUGCCGUGGCUGAUGA X GAA ACUGGUAGUU  929 CGUCCCUGUA CUGAUGA X GAA AUGCCGUGGG  931UGCGUCCCUG CUGAUGA X GAA AGAUGCCGUG  951 UGGAUGCAGA CUGAUGA X GAAAGCGGUCUUU  952 GUGGAUGCAG CUGAUGA X GAA AAGCGGUCUU  953 UGUGGAUGCACUGAUGA X GAA AAAGCGGUCU  959 AGAUGUUGUG CUGAUGA X GAA AUGCAGAAAG  968CCUGGUAGGA CUGAUGA X GAA AUGUUGUGGA  970 GCCCUGGUAG CUGAUGA X GAAAGAUGUUGUG  973 CCGGCCCUGG CUGAUGA X GAA AGGAGAUGUU  985 GGAGAAGGCGCUGAUGA X GAA ACCGGCCCUG  986 CGGAGAAGGC CUGAUGA X GAA AACCGGCCCU  991GUAGUCGGAG CUGAUGA X GAA AGGCGAACCG  992 GGUAGUCGGA CUGAUGA X GAAAAGGCGAACC  994 CGGGAUGUCG CUGAUGA X GAA AGAAGGCGAA 1000 CAGCUCCGGGCUGAUGA X GAA AGUCGGAGAA 1016 AUCUCUCCGG CUGAUGA X GAA AGGUUCAGCU 1027GGACGACUUG CUGAUGA X GAA AUCUCUCCGG 1028 AGGACGACUU CUGAUGA X GAAAAUCUCUCCG 1033 AUCGAAGGAC CUGAUGA X GAA ACUUGAAUCU 1036 GAAAUCGAAGCUGAUGA X GAA ACGACUUGAA 1039 GAUGAAAUCG CUGAUGA X GAA AGGACGACUU 1040CGAUGAAAUC CUGAUGA X GAA AAGGACGACU 1044 CCGUCGAUGA CUGAUGA X GAAAUCGAAGGAC 1045 GCCGUCGAUG CUGAUGA X GAA AAUCGAAGGA 1046 AGCCGUCGAUCUGAUGA X GAA AAAUCGAAGG 1049 CGUAGCCGUC CUGAUGA X GAA AUGAAAUCGA 1057GGGCUUCUCG CUGAUGA X GAA AGCCGUCGAU 1085 UCAUCCAGUU CUGAUGA X GAAAUCUUCCGGC 1106 CGGCCUCGAG CUGAUGA X GAA AUCCCGGCCU 1109 UGUCGGCCUCCUGAUGA X GAA AGGAUCCCGG 1124 UGACGGUGAG CUGAUGA X GAA ACCCUGUCGG 1127GGCUGACGGU CUGAUGA X GAA AGGACCCUGU 1133 AGUAGGGGCU CUGAUGA X GAAACGGUGAGGA 1141 CUCGGCGUAG CUGAUGA X GAA AGGGGCUGAC 1144 CUCCUCGGCGCUGAUGA X GAA AGUAGGGGCU 1157 UGCCGGAGAU CUGAUGA X GAA AGCUCCUCGG 1160CGAUGCCGGA CUGAUGA X GAA AUGAGCUCCU 1162 GGCGAUGCCG CUGAUGA X GAAAGAUGAGCUC 1169 AGCCCCUGGC CUGAUGA X GAA AUGCCGGAGA 1187 UGAUGUUCUGCUGAUGA X GAA AGCUCGCAGC 1196 UGAGGCGCAU CUGAUGA X GAA AUGUUGUCGA 1205UGAUGCCGGU CUGAUGA X GAA AGGCGCAUGA 1214 CGAUGCCGGU CUGAUGA X GAAAUGCCGGUGA 1223 UGCCGUUGAC CUGAUGA X GAA AUGCCGGUGA 1226 CCAUGCCGUUCUGAUGA X GAA ACGAUGCCGG 1241 CCCACUCGCU CUGAUGA X GAA ACGUCCAUGC 1270CACGGCGAUG CUGAUGA X GAA ACUUCUCCCU 1274 ACUUCACGGC CUGAUGA X GAAAUGUACUUGU 1285 CGACACGUCG CUGAUGA X GAA ACUUCACGGC 1294 CACGGCCGUCCUGAUGA X GAA ACACGUCGUA 1346 CCGGGAGCCC CUGAUGA X GAA ACCUCCGCCU 1352GGUCCACCGG CUGAUGA X GAA AGCCCGACCU 1370 CCACCAGCGG CUGAUGA X GAAAUGUUCCGGU 1384 CCUGCCGAUG CUGAUGA X GAA ACGCCACCAG 1385 GCCUGCCGAUCUGAUGA X GAA AACGCCACCA 1388 CCAGCCUGCC CUGAUGA X GAA AUGAACGCCA 1421CGGCCGCCAU CUGAUGA X GAA ACGUCGGGUC 1436 UGAGCUGCGG CUGAUGA X GAAAUGGCGGCCG 1445 CCAUCUCCAU CUGAUGA X GAA AGCUGCGGGA 1472 CCAGCAGAACCUGAUGA X GAA AUCUGCACGU 1475 UGCCCAGCAG CUGAUGA X GAA ACGAUCUGCA 1476GUGCCCAGCA CUGAUGA X GAA AACGAUCUGC 1501 CAUGCGCUCG CUGAUGA X GAAACUUCUUCUU 1502 GCAUGCGCUC CUGAUGA X GAA AACUUCUUCU 1514 CGGCGCUCAUCUGAUGA X GAA ACUUCUCCUC 1534 CUUGCCUGGG CUGAUGA X GAA ACUUCUCCUC 1535CCUUGCCUGG CUGAUGA X GAA AACUUCUCCU 1559 CGUUGAACUU CUGAUGA X GAAACCACGGCGC 1564 CGCCGCGUUG CUGAUGA C GAA ACUUGACCAC 1565 GCGCCGCGUUCUGAUGA X GAA AACUUGACCA 1589 CGCCGGCCAU CUGAUGA X GAA AUGUGGUGCG 1610UGGUGACGGC CUGAUGA C GAA AGCACGUCGG 1616 AGCGGCUGGU CUGAUGA X GAAACGGCGAGCA 1627 GCAGGGCUCG CUGAUGA X GAA AGCGGCUGGU 1628 CGCAGGGCUCCUGAUGA X GAA AAGCGGCUGG 1643 GCAGCUGGAU CUGAUGA X GAA AGGCCGCAGG 1646CCUGCAGCUG CUGAUGA X GAA AUGAGGCCGC 1666 GGGCGUUCCG CUGAUGA X GAAAUCGCAUCCC 1690 UCCACCGGUG CUGAUGA X GAA ACGCGCAGGC 1703 UGGUGUCGACCUGAUGA X GAA AGUCCACCGG 1706 UGAUGGUGUC CUGAUGA X GAA ACGAGUCCAC 1715UGCCUUCGAU CUGAUGA X GAA AUGGUGUCGA 1718 UCUUGCCUUC CUGAUGA X GAAAUGAUGGUGU 1735 GCCCAUGUGG CUGAUGA X GAA ACCCGGUCUU 1736 GGCCCAUGUGCUGAUGA X GAA AACCCGGUCU 1751 AGUCGACGCU CUGAUGA X GAA AGGCGGCCCA 1757CGUUGCAGUC CUGAUGA X GAA ACGCUGAGGC 1769 CCGGCUCCAC CUGAUGA X GAAACGUUGCAGU 1787 CCACCUUCUU CUGAUGA X GAA ACGUCCGCCG 1807 GGCGCGCUGCCUGAUGA X GAA AGGUGGUGGC 1820 CGACCACCUU CUGAUGA X GAA AUGGCGCGCU 1829CCGGCGUGCC CUGAUGA X GAA ACCACCUUGA 1843 CAUCUCCUCG CUGAUGA X GAAACGCCGGCGU 1871 AGAGAUCCUG CUGAUGA X GAA AUCAUGCAGU 1878 UUCCAGGAGACUGAUGA X GAA AUCCUGGAUC 1880 CCUUCCAGGA CUGAUGA X GAA AGAUCCUGGA 1882GCCCUUCCAG CUGAUGA X GAA AGAGAUCCUG 1922 CCCCGAGGCU CUGAUGA X GAAAGCAGCACGU 1928 CGGCGACCCC CUGAUGA X GAA AGGCUGAGCA 1934 CGCCGCCGGCCUGAUGA X GAA ACCCCGAGGC 1955 CCUCGCCUUC CUGAUGA X GAA ACCCCUGGCU 1970CGAGCGGCGC CUGAUGA X GAA AUCUCCUCGC 1979 UCUCCUUGGC CUGAUGA X GAAAGCGGCGCGA 2012 CUGCAGGCCG CUGAUGA X GAA ACUCUUCAGG 2013 CCUGCAGGCCCUGAUGA X GAA AACUCUUCAG 2033 CCACGCGCGA CUGAUGA X GAA AUCAGGGGGC 2035CACCACGCGC CUGAUGA X GAA AGAUCAGGGG 2055 AAGAUGUCCC CUGAUGA X GAAACAUGUUUGC 2063 UAUAUAAGAA CUGAUGA X GAA AUGUCCCAAC 2065 CAUAUAUAAGCUGAUGA X GAA AGAUGUCCCA 2066 GCAUAUAUAA CUGAUGA X GAA AAGAUGUCCC 2068CAGCAUAUAU CUGAUGA X GAA AGAAGAUGUC 2069 ACAGCAYAYA CYGAYGA X GAAAAGAAGAUGU 2071 AAACAGCAUA CUGAUGA X GAA AUAAGAAGAU 2073 CGAAACAGCACUGAUGA X GAA AUAUAAGAAG 2080 ACAUAAACGA CUGAUGA X GAA ACAGCAUAUA 2081CACAUAAACG CUGAUGA X GAA AACAGCAUAU 2082 UCACAUAAAC CUGAUGA X GAAAAACAGCAUA 2085 AUAUCACAUA CUGAUGA X GAA ACGAAACAGC 2086 CAUAUCACAUCUGAUGA X GAA AACGAAACAG 2087 CCAUAUCACA CUGAUGA X GAA AAACGAAACA 2094UACUUGUCCA CUGAUGA X GAA AUCACAUAAA 2104 CAGCUACACA CUGAUGA X GAAACUUGUCCAU 2110 AGCAAGCAGC CUGAUGA X GAA ACACAUACUU 2117 UAGCACAAGCCUGAUGA X GAA AGCAGCUACA 2121 ACACUAGCAC CUGAUGA X GAA AGCAAGCAGC 2127UAUAUUACAC CUGAUGA X GAA AGCACAAGCA 2132 UACACUAUAU CUGAUGA X GAAACACUAGCAC 2135 CACUACUCUA CUGAUGA X GAA AUUACACUAG 2137 ACCACUACACCUGAUGA X GAA AUAUUACACU 2142 UGGCCACCAC CUGAUGA X GAA ACACUAUAUU 2165AUGCGCUUAU CUGAUGA X GAA AGGUUGUGCC 2168 UUCAUGCGCU CUGAUGA X GAAAUUAGGUUGU 2181 CGCAAGCAAU CUGAUGA X GAA AGUUCAUGCG 2184 ACACGCAAGCCUGAUGA X GAA AUUAGUUCAU 2188 CUACACACGC CUGAUGA X GAA AGCAAUUAGU 2197GGUACUUAAC CUGAUGA X GAA ACACACGCAA 2200 AUCGGUACUU CUGAUGA X GAAACUACACACG 2201 GAUCGGUACU CUGAUGA X GAA AACUACACAC 2205 UACCGAUCGGCUGAUGA X GAA ACUUAACUAC 2211 UAAAAUUACC CUGAUGA X GAA AUCGGUACUU 2215AAUAUAAAAU CUGAUGA X GAA ACCGAUCGGU 2218 CGCAAUAUAA CUGAUGA X GAAAUUACCGAUC 2219 UCGCAAUAUA CUGAUGA X GAA AAUUACCGAU 2220 CUCGCAAUAUCUGAUGA X GAA AAAUUACCGA 2221 ACUCGCAAUA CUGAUGA X GAA AAAAUUACCG 2223UUACUCGCAA CUGAUGA X GAA AUAAAAUUAC 2225 AUUUACUCGC CUGAUGA X GAAAUAUAAAAUU 2232 UCCAUUUAUU CUGAUGA X GAA ACUCGCAAUA 2236 CAGGUCCAUUCUGAUGA X GAA AUUUACUCGC 2248 UUUCCACCAC CUGAUGA X GAA ACAGGUCCAU Where“X” represents stem II region of a HH ribozyme (Hertel et al., 1992Nucleic acids Res. 20 3252). The length of stem II may be ≧2 base-pairs.

TABLE IV HH Ribozyme Sequences Tested against GBSS mRNA nt. SequencePosition HH Ribozyme Sequence I.D.  425 CGACGAAGACCUGAUGAGGCCGAAAGGCCGAA ACGUUCAUGC  2  593 CUCCCAUCUUCUGAUGAGGCCGAAAGGCCGAA AUCUCGGACA  3  742 GUUGUCCCUGCUGAUGAGGCCGAAAGGCCGAA AGUCCGUUCC  4  812 GGUUGUUGUUCUGAUGAGGCCGAAAGGCCGAA AGGCUCAGAA  5  892 GAGGUAGCACCUGAUGAGGCCGAAAGGCCGAA AGAGAGGGCC  6  913 GUGGGACUGGCUGAUGAGGCCGAAAGGCCGAA AGUUGCUCUU  7  919 GAUGCCGUGGCUGAUGAGGCCGAAAGGCCGAA ACUGGUAGUU  8  953 UGUGGAUGCACUGAUGAGGCCGAAAGGCCGAA AAAGCGGUCU  9  959 AGAUGUUGUGCUGAUGAGGCCGAAAGGCCGAA AUGCAGAAAG 10  968 CCUGGUAGGACUGAUGAGGCCGAAAGGCCGAA AUGUUGUGGA 11 1016 AUCUCUCCGGCUGAUGAGGCCGAAAGGCCGAA AGGUUCAGCU 12 1028 AGGACGACUUCUGAUGAGGCCGAAAGGCCGAA AAUCUCUCCG 13 1085 UCAUCCAGUUCUGAUGAGGCCGAAAGGCCGAA AUCUUCCGGC 14 1187 UGAUGUUGUCCUGAUGAGGCCGAAAGGCCGAA AGCUCGCAGC 15 1196 UGAGGCGCAUCUGAUGAGGCCGAAAGGCCGAA AUGUUGUCGA 16 1226 CCAUGCCGUUCUGAUGAGGCCGAAAGGCCGAA ACGAUGCCGG 17 1241 CCCACUCGCUCUGAUGAGGCCGAAAGGCCGAA ACGUCCAUGC 18 1270 CACGGCGAUGCUGAUGAGGCCGAAAGGCCGAA ACUUGUCCCU 19 1352 GGUCCACCGGCUGAUGAGGCCGAAAGGCCGAA AGCCCGACCU 20 1421 CGGCCGCCAUCUGAUGAGGCCGAAAGGCCGAA ACGUCGGGUC 21 1534 CUUGCCUGGGCUGAUGAGGCCGAAAGGCCGAA ACUUCUCCUC 22 1715 UGCCUUCGAUCUGAUGAGGCCGAAAGGCCGAA AUGGUGUCGA 23 1787 CCACCUUCUUCUGAUGAGGCCGAAAGGCCGAA ACGUCCGCCG 24

TABLE V A GBSS Hairpin Ribozyme and Substrate Sequences nt. PositionHairpin Ribozyme Sequence Substrate  48 CUCCUGGC AGAA GUCGACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA CGACA GCC GCCAGGAG  129 CCCUGCCGAGAA GUGC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA GCACC GCC CGGCAGGG  468GUCGCCGA AGAA GCCG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA CGGCG GCCUCGGCGAC  489 CGGCGGCA AGAA GCCG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUACGGCG GCC UGCCGCCG  496 CCAUGGCC AGAA GCAGACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA CUGCC GCC GGCCAUGG  676 UCUCCAGGAGAA GUGG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA CCACU GUU CCUGGAGA  737UCCCUGUA AGAA GUUC ACCAGAGAAACACACGUUCUGGUACAUUACCUGGUA GAACG GACAUCAGGGA  760 GCAGGCUG AGAA GCAG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUACUGCG GUU CAGCCUGC 1298 GCCUCCAC AGAA GUCGACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA CGACG GCC GUGGAGGC 1427 GGGAUGGCAGAA GCCA ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA UGGCG GCC GCCAUCCC 1601GCGAGCAC AGAA GCGC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA GCGCC GACGUGCUCGC 1638 CUGGAUGA AGAA GCAG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUACUGCG GCC UCAUCCAG 1746 GACGCUGA AGAA GCCCACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA GGGCC GCC UCAGCGUC 1781 UUCUUGACAGAA GCCG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA CGGCG GAC GUCAAGAA 2077AUAAACGA AGAA GCAU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA AUGCU GUUUCGUUUAU

TABLE VB GBSS Hairpin Ribozyme and Substrate Sequences nt. PositionRibozyme Sequence Substrate  31 GUCGCCUC AGAA GGUGGUACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA ACCACCC GCC GAGGCGAC  48 CUCCUGGCAGAA GUCGCG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA CGCGACA GCC GCCAGGAG 105 GUGGACGG AGAA GUACAC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA GUGUACUGCU CCGUCCAC  110 CACUGGUG AGAA GAGCAGACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA CUGCUCC GUC CACCAGUG  129 CCCUGCCGAGAA GUGCGC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA GCGCACC GCC CGGCAGGG 142 ACGAGAUG AGAA GCCCUG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA CAGGGCUGCU CAUCUCGU  182 GUGGCUAG AGAA GCCUAGACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA CAUGGCG GCU CUAGCCAC  199 UUGCGACGAGAA GCGACG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA CGUCGCA GCU CGUCGCAA 219 GACGCCCA AGAA GGCGCG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA CGCGCCGGCC UGGGCGUC  233 GUGGACGC AGAA GGGACGACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA CGUCCCG GAC GCGUCCAC  249 GGCGCCGCAGAA GAACGU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA ACGUUCC GCC GCGGCGCC 283 CCGACGCC AGAA GGCCCC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA GGGGCCGGAC GGCGUCGG  316 GCGCGCUG AGAA GAAUGCACGAGAGAAACACACGUUGUGGUACAUUACCUGGUA GCAUUCG GAC CAGCGCGC  388 CGACGAGCAGAA GGAACC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA GGUUCCC GUC GCUCGUCG 468 GUCGCCGA AGAA GCCGGU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA ACCGGCGGCC ACGGCGAC  489 CGGCGGCA AGAA GCCGAGACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA CUCGGCG GCC UGCCGCCG  493 UGGCCGGCAGAA GGCCGC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA GCGGCCU GCC GCCGGCCA 496 CCAUGGCC AGAA GCAGGC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA GCCUGCCGCC GGCCAUGG  676 UCUCCAGG AGAA GUGGGUACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA ACCCACU GUU CCUGGAGA  725 GUUCCAGCAGAA GGCCCG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA CGGGCCU GAC GCUGGAAC 737 UCCCUGUA AGAA GUUCCA ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA UGGAACGGAC AUCAGGGA  754 UGAACCGC AGAA GGUUGUACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA ACAACCA GCU GCGGUUCA  760 GCAGGCUGAGAA GCAGCU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA AGCUGCG GUU CAGCCUGC 765 GCAUAGCA AGAA GAACCG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA CGGUUCAGCC UGCUAUGC  834 CCCGUAUG AGAA GGAGAAACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA UUCUCCG GAC CAUACGGG  882 CGAGAGAGAGAA GGUGUG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA CACACCG GCC CUCUCUCG 916 UGCCGUGG AGAA GGUAGU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA ACUACCAGUC CCACGGCA  947 AUGCAGAA AGAA GUCUUUACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA AAAGACC GCU UUCUGCAU  982 AGAAGGCGAGAA GGCCCU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA AGGGCCG GUU CGCCUUCU 995 UCCGGGUA AGAA GAGAAG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA CUUCUCCGAC UACCCGGA 1134 GUAGUAGG AGAA GACGGUACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA ACCGUCA GCC CCUACUAC 1298 GCCUCCACAGAA GUCGAC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA GUCGACG GCC GUGGAGGC1372 ACGCCACC AGAA GGAUGU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA ACAUCCCGCU GGUGGCGU 1415 GCCAUGAC AGAA GGUCCCACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA GGGACCC GAC GUCAUGGC 1427 GGGAUGGCAGAA GCCAUG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA CAUGGCG GCC GCCAUCCC1441 UCUCCAUG AGAA GCGGGA ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA UCCCGCAGCU CAUGGAGA 1468 GCAGAACG AGAA GCACGUACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA ACGUGCA GAU CGUUCUGC 1477 CCGUGCCCAGAA GAACGA ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA UCGUUCU GCU GGGCACGG1601 GCGAGCAC AGAA GCGCCG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA CGGCGCCGAC GUGCUCGC 1620 CUCGAAGC AGAA GGUGACACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA GUCACCA GCC GCUUCGAG 1623 GGGCACGAAGAA GCUGGU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA ACCAGCC GCU UCGAGCCC1638 CUGGAUGA AGAA GCAGGG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA CCCUGCGGCC UCAUCCAG 1648 UCCCCUGC AGAA GGAUGAACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA UCAUCCA GCU GCAGGGGA 1746 GACGCUGAAGAA GCCCAU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA AUGGGCC GCC UCAGCGUC1781 UUCUUGAC AGAA GCCGGC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA GCCGGCGGAC GUCAAGAA 1918 CGAGGCUG AGAA GCACGUACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA ACGUGCU GCU CAGCCUCG 1923 GACCCCGAAGAA GAGCAG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA CUGCUCA GCC UCGGGGUC1975 CCUUGGCG AGAA GCGCGA ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA UCGCGCCGCU CGCCAAGG 2014 GGCCUGCA AGAA GAACUCACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA GAGUUCG GCC UGCAGGCC 2029 CGCGCGAGAGAA GGGGGC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA GCCCCCU GAU CUCGCGCG2077 AUAAACGA AGAA GCAUAU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA AUAUGCUGUU UVHUUUAU 2113 CACAAGCA AGAA GCUACAACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA UGUAGCU GCU UGCUUGUG 2207 AAUUACCGAGAA GUACUU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA AAGUACC GAU CGGUAAUU

TABLE VI Delta-9 Desaturase HH Ribozyme Target Sequences nt. PositionSubstrate  13 CGCGCCCUC UGCCGCUU  21 CUGCCGCUU GUUCGUUC  24 CCGCUUGUUCGUUCCUC  25 CGCUUGUUC GUUCCUCG  28 UUGUUCGUU CCUCGCGC  29 UGUUCGUUCCUCGCGCU  32 UCGUUCCUC GCGCUCGC  38 CUCGCGCUC GCCACCAG  63 ACACACAUCCCAAUCUC  69 AUCCCAAUC UCGCGAGG  71 CCCAAUCUC GCGAGGGC  92 AGCAGGGUCUGCGGCGG  117 GCCGCGCUU CCGGCUCC  118 CCGCGCUUC CGGCUCCC  124 UUCCGGCUCCCCUUCCC  129 GCUCCCCUU CCCAUUGG  130 CUCCCCUUC CCAUUGGC  135 CUUCCCAUUGGCCUCCA  141 AUUGGCCUC CACGAUGG  154 AUGGCGCUC CGCCUCAA  160 CUCCGCCUCAACGACGU  169 AACGACGUC GCGCUCUG  175 GUCGCGCUC UGCCUCUC  181 CUCUGCCUCUCCCCGCC  183 CUGCCUCUC CCCGCCGC  193 CCGCCGCUC GCCGCCCG  228 CGGCAGGUUCGUCGCCG  229 GGCAGGUUC GUCGCCGU  232 AGGUUCGUC GCCGUCGC  238 GUCGCCGUCGCCUCCAU  243 CGUCGCCUC CAUGACGU  252 CAUGACGUC CGCCGUCU  259 UCCGCCGUCUCCACCAA  261 CGCCGUCUC CACCAAGG  271 ACCAAGGUC GAGAAUAA  278 UCGAGAAUAAGAAGCCA  288 GAAGCCAUU UGCUCCUC  289 AAGCCAUUU GCUCCUCC  293 CAUUUGCUCCUCCAAGG  296 UUGCUCCUC CAAGGGAG  307 AGGGAGGUA CAUGUCCA  313 GUACAUGUCCAGGUUAC  319 GUCCAGGUU ACACAUUC  320 UCCAGGUUA CACAUUCA  326 UUACACAUUCAAUGCCA  327 UACACAUUC AAUGCCAC  338 UGCCACCUC ACAAGAUU  346 CACAAGAUUGAAAUUUU  352 AUUGAAAUU UUCAAGUC  353 UUGAAAUUU UCAAGUCG  354 UGAAAUUUUCAAGUCGC  355 GAAAUUUUC AAGUCGCU  360 UUUCAAGUC GCUUGAUG  364 AAGUCGCUUGAUGAUUG  371 UUGAUGAUU GGGCAUGA  377 AUUGGGCUA GAGAUAAU  383 CUAGAGAUAAUAUCUUG  386 GAGAUAAUA UCUUGACG  388 GAUAAUAUC UUGACGCA  390 UAAUAUCUUGACGCAUC  398 UGACGCAUC UCAAGCCA  400 ACGCAUCUC AAGCCAGU  409 AAGCCAGUCGAGAAGUG  419 AGAAGUGUU GGCAGCCA  434 CACAGGAUU UCCUCCCG  435 ACAGGAUUUCCUCCCGG  436 CAGGAUUUC CUCCCGGA  439 GAUUUCCUC CCGGACCC  453 CCCAGCAUCUGAAGGAU  462 UGAAGGAUU UCAUGAUG  463 GAAGGAUUU CAUGAUGA  464 AAGGAUUUCAUGAUGAA  475 GAUGAAGUU AAGGAGCU  476 AUGAAGUUA AGGAGCUC  484 AAGGAGCUCAGAGAACG  505 AAGGAAAUC CCUGAUGA  515 CUGAUGAUU AUUUUGUU  516 UGAUGAUUAUUUUGUUU  518 AUGAUUAUU UUGUUUGU  519 UGAUUAUUU UGUUUGUU  520 GAUUAUUUUGUUUGUUU  523 UAUUUUGUU UGUUUGGU  524 AUUUUGUUU GUUUGGUG  527 UUGUUUGUUUGGUGGGA  528 UGUUUGUUU GGUGGGAG  544 GACAUGAUU ACCGAGGA  545 ACAUGAUUACCGAGGAA  557 AGGAAGCUC UACCAACA  559 GAAGCUCUA CCAACAUA  567 ACCAACAUACCAGACUA  575 ACCAGACUA UGCUUAAC  580 ACUAUGCUU AACACCCU  581 CUAUGCUUAACACCCUC  589 AACACCCUC GACGGUGU  598 GACGGUGUC AGAGAUGA  637 UGGGCUGUUUGGACGAG  638 GGGCUGUUU GGACGAGG  680 AUGGUGAUC UGCUCAAC  685 CAACAAGUAUAUGUACC  693 CAACAAGUA UAUGUACC  695 ACAAGUAUA UGUACCUC  699 GUAUAUGUACCUCACUG  703 AUGUACCUC ACUGGGAG  719 GGGUGGAUA UGAGGCAG  730 AGGCAGAUUGAGAAGAC  742 AAGACAAUU CAGUAUCU  743 AGACAAUUC AGUAUCUU  747 AAUUCAGUAUCUUAUUG  749 UUCAGUAUC UUAUUGGC  751 CAGUAUCUU AUUGGCUC  752 AGUAUCUUAUUGGCUCU  754 UAUCUUAUU GGCUCUGG  759 UAUUGGCUC UGGAAUGG  770 GAAUGGAUCCUAGGACU  773 UGGAUCCUA GGACUGAG  785 CUGAGAAUA AUCCUUAU  788 AGAAUAAUCCUUAUCUU  791 AUAAUCCUU AUCUUGGU  792 UAAUCCUUA UCUUGGUU  794 AUCCUUAUCUUGGUUUC  796 CCUUAUCUU GGUUUCAU  800 AUCUUGGUU UCAUCUAC  801 UCUUGGUUUCAUCUACA  802 CUUGGUUUC AUCUACAC  805 GGUUUCAUC UACACCUC  807 UUUCAUCUACACCUCCU  813 CUACACCUC CUUCCAAG  816 CACCUCCUU CCAAGAGC  817 ACCUCCUUCCAAGAGCG  834 GGCGACCUU CAUCUCAC  835 GCGACCUUC AUCUCACA  838 ACCUUCAUCUCACACGG  840 CUUCAUCUC ACACGGGA  857 ACACUGCUC GUCACGCC  860 CUGCUCGUCACGCCAAG  873 CAAGGACUU UGGCGACU  874 AAGGACUUU GGCGACUU  882 UGGCGACUUAAAGCUUG  883 GGCGACUUA AAGCUUGC  889 UUAAAGCUU GCACAAAU  898 GCACAAAUCUGCGGCAU  907 UGCGGCAUC AUCGCCUC  910 GGCAUCAUC GCCACAGA  915 CAUCGCCUCAGAUGAGA  942 AACUGCGUA CACCAAGA  952 ACCAAGAUC GUGGAGAA  966 GAAGCUGUUUGAGAUCG  967 AAGCUGUUU GAGAUCGA  973 UUUGAGAUC GACCCUGA  986 CUGAUGGUACCGUGGUC  994 ACCGUGGUC GCUCUGGC  998 UGGUCGCUC UGGCUGAC 1024 AAGAAGAUCUCAAUGCC 1026 GAAGAUCUC AAUGCCUG 1047 CCUGAUGUU UGACGGGC 1048 CUGAUGUUUGACGGGCA 1071 CAAGCUGUU CGAGCACU 1072 AAGCUGUUC GAGCACUU 1080 CGAGCACUUCUCCAUGG 1081 GAGCACUUC UCCAUGGU 1083 GCACUUCUC CAUGGUCG 1090 UCCAUGGUCGCGCAGAG 1102 CAGAGGCUU GGCGUUUA 1108 CUUGGCGUU UACACCGC 1109 UUGGCGUUUACACCGCC 1110 UGGCGUUUA CACCGCCA 1125 CAGGGACUA CGCCGACA 1135 GCCGACAUCCUCGAGUU 1138 GACAUCCUC GAGUUCCU 1143 CCUCGAGUU CCUCGUCG 1144 CUCGAGUUCCUCGUCGA 1147 GAGUUCCUC GUCGACAG 1150 UUCCUCGUC GACAGGUG 1181 UGACUGGUCUGUCGGGU 1185 UGGUCUGUC GGGUGAAG 1212 GCAGGACUA CCUUUGCA 1216 GACUACCUUUGCACCCU 1217 ACUACCUUU GCACCCUU 1225 UGCACCCUU GCUUCAAG 1229 CCCUUGCUUCAAGAAUC 1230 CCUUGCUUC AAGAAUCA 1237 UCAAGAAUC AGGAGGCU 1292 CGCUGCCUUUCAGCUGG 1293 GCUGCCUUU CAGCUGGG 1294 CUGCCUUUC AGCUGGGU 1303 AGCUGGGUAUACGGUAG 1305 CUGGGUAUA CGGUAGGG 1310 UAUACGGUA GGGACGUC 1318 AGGGACGUCCAACUGUG 1331 UGUGAGAUC GGAAACCU 1348 GCUGCGGUC UGCUUAGA 1353 GGUCUGCUUAGACAAGA 1354 GUCUGCUUA GACAAGAC 1372 UGCUGUGUC UGCGUUAC 1378 GUCUGCGUUACAUAGGU 1379 UCUGCGUUA CAUAGGUC 1383 CGUUACAUA GGUCUCCA 1387 ACAUAGGUCUCCAGGUU 1389 AUAGGUCUC CAGGUUUU 1395 CUCCAGGUU UUGAUCAA 1396 UCCAGGUUUUGAUCAAA 1397 CCAGGUUUU GAUCAAAU 1401 GUUUUGAUC AAAUGGUC 1409 CAAAUGGUCCCGUGUCG 1416 UCCCGUGUC GUCUUAUA 1419 CGUGUCGUC UUAUAGAG 1421 UGUCGUCUUAUAGAGCG 1422 GUCGUCUUA UAGAGCGA 1424 CGUCUUAUA GAGCGAUA 1432 AGAGCGAUAGGAGAACG 1444 GAACGUGUU GGUCUGUG 1448 GUGUUGGUC UGUGGUGU 1457 UGUGGUGUAGCUUUGUU 1461 GUGUAGCUU UGUUUUUA 1462 UGUAGCUUU GUUUUUAU 1465 AGCUUUGUUUUUAUUUU 1466 GCUUUGUUU UUAUUUUG 1467 CUUUGUUUU UAUUUUGU 1468 UUUGUUUUUAUUUUGUA 1469 UUGUUUUUA UUUUGUAU 1471 GUUUUUAUU UUGUAUUU 1472 UUUUUAUUUUGUAUUUU 1473 UUUUAUUUU GUAUUUUU 1476 UAUUUUGUA UUUUUCUG 1478 UUUUGUAUUUUUGUGCU 1479 UUUGUAUUU UUCUGCUU 1480 UUGUAUUUU UVUGCUUU 1481 UGUAUUUUUCUGCUUUG 1482 GUAUUUUUC UGCUUUGA 1487 UUUCUGCUU UGAUGUAC 1488 UUCUGCUUUGAUGAUCA 1494 UUUGAUGUA CAACCUGU 1546 CAUGCCGUA CUUUGUCU 1549 GCCGUACUUUGUCUGUC 1550 CCGUACUUU GUCUGUCG 1553 UACUUUGUC UGUCGCUG 1557 UUGUCUGUCGCUGGCGG 1571 CGGUGUGUU UCGGUAUG 1572 GGUGUGUUU CGGUAUGU 1573 GUGUGUUUCGGUAUGUU 1577 GUUUCGGUA UGUUAUUU 1581 CGGUAUGUU AUUUGAGU 1582 GGUAUGUUAUUUGAGUU 1584 UAUGUUAUU UGAGUUGC 1585 AUGUUAUUU GAGUUGCU 1590 AUUUGAGUUGCUCAGAU 1594 GAGUUGCUC AGAUCUGU 1599 GCUCAGAUC UGUUAAAA 1603 AGAUCUGUUAAAAAAAA 1604 GAUCUGUUA AAAAAAAA

TABLE VII Delta-9 Desaturase HH Ribozyme Sequences nt. Position Ribozymesequence 13 AAGCGGCA CUGAUGA X GAA AGGGCGCG 21 GAACGAAC CUGAUGA X GAAAGCGGCAG 24 GAGGAACG CUGAUGA X GAA ACAAGCGG 25 CGAGGAAC CUGAUGA X GAAAACAAGCG 28 GCGCGAGG CUGAUGA X GAA ACGAACAA 29 AGCGCGAG CUGAUGA X GAAAACGAACA 32 GCGAGCGC CUGAUGA X GAA AGGAACGA 38 CUGGUGGC CUGAUGA X GAAAGCGCGAG 63 GAGAUUGG CUGAUGA X GAA AUGUGUGU 69 CCUCGCGA CUGAUGA X GAAAUUGGGAU 71 GCCCUCGC CUGAUGA X GAA AGAUUGGG 92 CCGCCGCA CUGAUGA X GAAACCCUGCU 117 GGAGCCGG CUGAUGA X GAA AGCGCGGC 118 GGGAGCCG CUGAUGA X GAAAAGCGCGG 124 GGGAAGGG CUGAUGA X GAA AGCCGGAA 129 CCAAUGGG CUGAUGA X GAAAGGGGAGC 130 GCCAAUGG CUGAUGA X GAA AAGGGGAG 135 UGGAGGCC CUGAUGA X GAAAUGGGAAG 141 CCAUCGUG CUGAUGA X GAA AGGCCAAU 154 UUGAGGCG CUGAUGA X GAAAGCGCCAU 160 ACGUCGUU CUGAUGA X GAA AGGCGGAG 169 CAGAGCGC CUGAUGA X GAAACGUCGUU 175 GAGAGGCA CUGAUGA X GAA AGCGCGAC 181 GGCGGGGA CUGAUGA X GAAAGGCAGAG 183 GCGGCGGG CUGAUGA X GAA AGAGGCAG 193 CGGGCGGC CUGAUGA X GAAAGCGGCGG 228 CGGCGACG CUGAUGA X GAA ACCUGCCG 229 ACGGCGAC CUGAUGA X GAAAACCUGCC 232 GCGACGGC CUGAUGA X GAA ACGAACCU 238 AUGGAGGC CUGAUGA X GAAACGGCGAC 243 ACGUCAUG CUGAUGA X GAA AGGCGACG 252 AGACGGCG CUGAUGA X GAAACGUCAUG 259 UUGGUGGA CUGAUGA X GAA ACGGCGGA 261 CCUUGGUG CUGAUGA X GAAAGACGGCG 271 UUAUUCUC CUGAUGA X GAA ACCUUGGU 278 UGGCUUCU CUGAUGA X GAAAUUCUCGA 288 GAGGAGCA CUGAUGA X GAA AUGGCUUC 289 GGAGGAGC CUGAUGA X GAAAAUGGCUU 293 CCUUGGAG CUGAUGA X GAA AGCAAAUG 296 CUCCCUUG CUGAUGA X GAAAGGAGCAA 307 UGGACAUG CUGAUGA X GAA ACCUCCCU 313 GUAACCUG CUGAUGA X GAAACAUGUAC 319 GAAUGUGU CUGAUGA X GAA ACCUGGAC 320 UGAAUGUG CUGAUGA X GAAAACCUGGA 326 UGGCAUUG CUGAUGA X GAA AUGUGUAA 327 GUGGCAUU CUGAUGA X GAAAAUGUGUA 338 AAUCUUGU CUGAUGA X GAA AGGUGGCA 346 AAAAUUUC CUGAUGA X GAAAUCUUGUG 352 GACUUGAA CUGAUGA X GAA AUUUCAAU 353 CGACUUGA CUGAUGA X GAAAAUUUCAA 354 GCGACUUG CUGAUGA X GAA AAAUUUCA 355 AGCGACUU CUGAUGA X GAAAAAAUUUC 360 CAUCAAGC CUGAUGA X GAA ACUUGAAA 364 CAAUCAUC CUGAUGA X GAAAGCGACUU 371 UCUAGCCC CUGAUGA X GAA AUCAUCAA 377 AUUAUCUC CUGAUGA X GAAAGCCCAAU 383 CAAGAUAU CUGAUGA X GAA AUCUCUAG 386 CGUCAAGA CUGAUGA X GAAAUUAUCUC 388 UGCGUCAA CUGAUGA X GAA AUAUUAUC 390 GAUGCGUC CUGAUGA X GAAAGAUAUUA 398 UGGCUUGA CUGAUGA X GAA AUGCGUCA 400 ACUGGCUU CUGAUGA X GAAAGAUGCGU 409 CACUUCUC CUGAUGA X GAA ACUGGCUU 419 UGGCUGCC CUGAUGA X GAAACACUUCU 434 CGGGAGGA CUGAUGA X GAA AUCCUGUG 435 CCGCGAGG CUGAUGA X GAAAAUCCUGU 436 UCCGGGAG CUGAUGA X GAA AAAUCCUG 439 GGGUCCGG CUGAUGA X GAAAGGAAAUC 453 AUCCUUCA CUGAUGA X GAA AUGCUGGG 462 CAUCAUGA CUGAUGA X GAAAUCCUUCA 463 UCAUCAUG CUGAUGA X GAA AAUCCUUC 464 UUCAUCAU CUGAUGA X GAAAAAUCCUU 475 AGCUCCUU CUGAUGA X GAA ACUUCAUC 476 GAGCUCCU CUGAUGA X GAAAACUUCAU 484 CGUUCUCU CUGAUGA X GAA AGCUCCUU 505 UCAUCAGG CUGAUGA X GAAAUUUCCUU 515 AACAAAAU CUGAUGA X GAA AUCAUCAG 516 AAACAAAA CUGAUGA X GAAAAUCAUCA 518 ACAAACAA CUGAUGA X GAA AUAAUCAU 519 AACAAACA CUGAUGA X GAAAAUAAUCA 520 AAACAAAC CUGAUGA X GAA AAAUAAUC 523 ACCAAACA CUGAUGA X GAAACAAAAUA 524 CACCAAAC CUGAUGA X GAA AACAAAAU 527 UCCCACCA CUGAUGA X GAAACAAACAA 528 CUCCCACC CUGAUGA X GAA AACAAACA 544 UCCUCGGU CUGAUGA X GAAAUCAUGUC 545 UUCCUCGG CUGAUGA X GAA AAUCAUGU 557 UGUUGGUA CUGAUGA X GAAAGCUUCCU 559 UAUCUUGG CUGAUGA X GAA AGAGCUUC 567 UAGUCUGG CUGAUGA X GAAAUGUUGGU 575 GUUAAGCA CUGAUGA X GAA AGUCUGGU 580 AGGGUGUU CUGAUGA X GAAAGCAUAGU 581 GAGGGUGU CUGAUGA X GAA AAGCAUAG 589 ACACCGUC CUGAUGA X GAAAGGGUGUU 598 UCAUCUCU CUGAUGA X GAA ACACCGUC 637 CUCGUCCA CUGAUGA X GAAACAGCCCA 638 CCUCGUCC CUGAUGA X GAA AACAGCCC 680 GUUGAGCA CUGAUGA X GAAAUCACCAU 685 UAGUUGUU CUGAUGA X GAA AGCAGAUC 693 GGUACAUA CUGAUGA X GAAACUUGUUG 695 GAGGUACA CUGAUGA X GAA AUACUUGU 699 CAGUGAGG CUGAUGA X GAAACAUAUAC 703 CUCCCAGU CUGAUGA X GAA AGGUACAU 719 CUGCCUCA CUGAUGA X GAAAUCCACCC 730 GUCUUCUC CUGAUGA X GAA AUCUGCCU 742 AGAUACUG CUGAUGA X GAAAUUGUCUU 743 AAGAUACU CUGAUGA X GAA AAUUGUCU 747 CAAUAAGA CUGAUGA X GAAACUGAAUU 749 GCCAAUAA CUGAUGA X GAA AUACUGAA 751 GAGCCAAU CUGAUGA X GAAAGAUACUG 752 AGAGCCAA CUGAUGA X GAA AAGAUACU 754 CCAGAGCC CUGAUGA x GAAAUAAGAUA 759 CCAUUCCA CUGAUGA X GAA AGCCAAUA 770 AGUCCUAG CUGAUCA X GAAAUCCAUUC 773 CUCAGUCC CUGAUGA X GAA AGGAUCCA 785 AUAAGGAU CUGAUGA X GAAAUUCUCAG 788 AAGAUAAG CUGAUGA X GAA AUUAUUCU 791 ACCAAGAU CUGAUGA X GAAAGGAUUAU 792 AACCAAGA CUGAUGA X GAA AAGGAUUA 794 GAAACCAA CUGAUGA X GAAAUAAGGAU 796 AUGAAACC CUGAUGA X GAA AGAUAAGG 800 GUAGAUGA CUGAUGA X GAAACCAAGAU 801 UGUAGAUG CUGAUGA X GAA AACCAAGA 802 GUGUAGAU CUGAUGA X GAAAAAGCAAG 805 GAGGUGUA CUGAUGA X GAA AUGAAACC 807 AGGAGGUG CUGAUGA X GAAAGAUGAAA 813 CUUGGAAG CUGAUGA X GAA AGGUGUAG 816 GCUCUUGG CUGAUGA X GAAAGGAGGUG 817 CGCUCUUG CUGAUGA X GAA AAGGAGGU 834 GUGAGAUG CUGAUGA X GAAAGGUCGCC 835 UGUGAGAU CUGAUGA X GAA AAGGUCGC 838 CCGUGUGA CUGAUGA X GAAAUGAAGGU 840 UCCCGUGU CUGAUGA X GAA AGAUGAAG 857 GGCGUGAC CUGAUGA X GAAAGCAGUGU 860 CUUGGCGU CUGAUGA X GAA ACGAGCAG 873 AGUCGCCA CUGAUGA X GAAAGUGCUUG 874 AAGUCGCC CUGAUGA X GAA AAGUCCUU 882 CAAGCUUU CUGAUGA X GAAAGUCGCCA 883 GCAAGCUU CUGAUGA X GAA AAGUCGCC 889 AUUUGUGC CUGAUGA X GAAAGCUUUAA 898 AUGCCGCA CUGAUGA X GAA AUUUGUGC 907 GAGGCGAU CUGAUGA X GAAAUGCCGCA 910 UCUGAGGC CUGAUGA X GAA AUGAUGCC 915 UCUCAUCU CUGAUGA X GAAAGGCGAUG 942 UCUUGGUG CUGAUGA X GAA ACGCAGUU 952 UUCUCCAC CUGAUGA X GAAAUCUUGGU 966 CGAUCUCA CUGAUGA X GAA ACAGCUUC 967 UCGAUCUC CUGAUGA X GAAAACAGCUU 973 UCAGGGUC CUGAUGA X GAA AUCUCAAA 986 GACCACGG CUGAUGA X GAAACCAUCAG 994 GCCAGAGC CUGAUGA X GAA ACCACGGU 998 GUCAGCCA CUGAUGA X GAAAGCGACCA 1024 GGCAUUGA CUGAUGA X GAA AUCUUCUU 1026 CAGGCAUU CUGAUGA XGAA AGAUCUUC 1047 GCCCGUCA CUGAUGA X GAA ACAUCAGG 1048 UGCCCGUC CUGAUGAX GAA AACAUCAG 1071 AGUGCUCG CUGAUGA X GAA ACAGCUUG 1072 AAGUGCUCCUGAUGA X GAA AACAGCUU 1080 CCAUGGAG CUGAUGA X GAA AGUGCUCG 1081ACCAUGGA CUGAUGA X GAA AAGUGCUC 1083 CGACCAUG CUGAUGA X GAA AGAAGUGC1090 CUCUGCGC CUGAUGA X GAA ACCAUGGA 1102 UAAACGCC CUGAUGA X GAAAGCCUCUG 1108 GCGGUGUA CUGAUGA X GAA ACGCCAAG 1109 GGCGGUGU CUGAUGA XGAA AACGCCAA 1110 UGGCGGUG CUGAUGA X GAA AAACGCCA 1125 UGUCGGCG CUGAUGAX GAA AGUCCCUG 1135 AACUCGAG CUGAUGA X GAA AUGUCGGC 1138 AGGAACUCCUGAUGA X GAA AGGAUGUC 1143 CGACGAGG CUGAUGA X GAA ACUCCAGG 1144UCGACGAG CUGAUGA X GAA AACUCGAG 1147 CUGUCGAC CUGAUGA X CAA AGGAACUC1150 CACCUGUC CUGAUGA X GAA ACGAGGAA 1181 ACCCGACA CUGAUGA X GAAACCAGUCA 1185 CUUCACCC CUGAUGA X GAA ACAGACCA 1212 UGCAAAGG CUGAUGA XGAA AGUCCUGC 1216 AGGGUGCA CUGAUGA X GAA AGGUAGUC 1217 AAGGGUGC CUGAUGAX GAA AAGGUAGU 1225 CUUGAAGC CUGAUGA X GAA AGGGUGCA 1229 GAUUCUUGCUGAUGA X GAA AGCAAGGG 1230 UGAUUCUU CUGAUGA X GAA AAGCAAGG 1237AGCCUCCU CUGAUGA X GAA AUUCUUGA 1292 CCAGCUGA CUGAUGA X GAA AGGCAGCG1293 CCCAGCUG CUGAUGA X GAA AAGGCAGC 1294 ACCCAGCU CUGAUGA X GAAAAAGGCAG 1303 CUACCGUA CUGAUGA X GAA ACCCAGCU 1305 CCCUACCG CUGAUGA XGAA AUACCCAG 1310 GACGUCCC CUGAUGA X GAA ACCGUAUA 1318 CACAGUUG CUGAUGAX GAA ACGUCCCU 1331 AGGUUUCC CUGAUGA X GAA AUCUCACA 1348 UCUAAGCACUGAUGA X GAA ACCGCAGC 1353 UCUUGUCU CUGAUGA X GAA AGCAGACC 1354GUCUUGUC CUGAUGA X GAA AAGCAGAC 1372 GUAACGCA CUGAUGA X GAA ACACAGCA1378 ACCUAUGU CUGAUGA X GAA ACGCAGAC 1379 GACCUAUG CUGAUGA x GAAAACGCAGA 1383 UGGAGACC CUGAUGA X GAA AUGUAACG 1387 AACCUGGA CUGAUGA XGAA ACCUAUGU 1389 AAAACCUG CUGAUGA X GAA AGACCUAU 1395 UUGAUCAA CUGAUGAX GAA ACCUGGAG 1396 UUUGAUCA CUGAUGA X GAA AACCUGGA 1397 AUUUGAUCCUGAUGA X GAA AAACCUGG 1401 GACCAUUU CUGAUGA X GAA AUCAAAAC 1409CGACACGG CUGAUGA X GAA ACCAUUUG 1416 UAUAAGAC CUGAUGA X GAA ACACGGGA1419 CUCUAUAA CUGAUGA x GAA ACGACACG 1421 CGCUCUAU CUGAUGA X GAAAGACGACA 1422 UCGCUCUA CUGAUGA X GAA AAGACGAC 1424 UAUCGCUC CUGAUGA xGAA AUAAGACG 1432 CGUUCUCC CUGAUGA X GAA AUCGCUCU 1444 CACAGACC CUGAUGAX GAA ACACGUUC 1448 ACACCACA CUGAUGA X GAA ACCAACAC 1457 AACAAAGCCUGAUGA X GAA ACACCACA 1461 UAAAAACA CUGAUGA X GAA AGCUACAC 1462AUAAAAAC CUGAUGA X GAA AAGCUACA 1465 AAAAUAAA CUGAUGA X GAA ACAAAGCU1466 CAAAAUAA CUGAUGA X GAA AACAAAGC 1467 ACAAAAUA CUGAUGA X GAAAAACAAAG 1468 UACAAAAU CUGAUGA X GAA AAAACAAA 1469 AUACAAAA CUGAUGA XGAA AAAAACAA 1471 AAAUACAA CUGAUGA X GAA AUAAAAAC 1472 AAAAUACA CUGAUGAX GAA AAUAAAAA 1473 AAAAAUAC CUGAUGA X GAA AAAUAAAA 1476 CAGAAAAACUGAUGA X GAA ACAAAAUA 1478 AGCAGAAA CUGAUGA X GAA AUACAAAA 1479AAGCAGAA CUGAUGA X GAA AAUACAAA 1480 AAAGCAGA CUGAUGA X GAA AAAUACAA1481 CAAAGCAG CUGAUGA X GAA AAAAUACA 1482 UCAAAGCA CUGAUGA X GAAAAAAAUAC 1487 GUACAUCA CUGAUGA X GAA AGCAGAAA 1488 UGUACAUC CUGAUGA XGAA AAGCAGAA 1494 ACAGGUUG CUGAUGA X GAA ACAUCAAA 1546 AGACAAAG CUGAUGAX GAA ACGGCAUG 1549 GACAGACA CUGAUGA X GAA AGUACGGC 1550 CGACAGACCUGAUGA X GAA AAGUACGG 1553 CAGCGACA CUGAUGA X GAA ACAAAGUA 1557CCGCCAGC CUGAUGA X GAA ACAGAGAA 1571 CAUACCGA CUGAUGA X GAA ACACACCG1572 ACAUACCG CUGAUGA X GAA AACACACC 1573 AACAUACC CUGAUGA X GAAAAACACAC 1577 AAAUAACA CUGAUGA X GAA ACCGAAAC 1581 ACUCAAAU CUGAUGA XGAA ACAUACCG 1582 AACUCAAA CUGAUGA X GAA AACAUACC 1584 GCAACUCA CUGAUGAX GAA AUAACAUA 1585 AGCAACUC CUGAUGA X GAA AAUAACAU 1590 AUCUGAGCCUGAUGA X GAA ACUCAAAU 1594 ACAGAUCU CUGAUGA X GAA AGCAACUC 1599UUUUAACA CUGAUGA X GAA AUCUGAGC 1603 UUUUUUUU CUGAUGA X GAA ACAGAUCU1604 UUUUUUUU CUGAUGA X GAA AACAGAUC Where “X” represents stem II regionof a HH ribozyme (Hertel et al., 1992 Nucleic Acids Res. 20 3252). Thelength of stem II may be ≧2 base-pairs.

TABLE VIII Delta-9 Desaturase Hairpin Ribozyme and Substrate Sequencesnt. Position Ribozyme Substrate 14 GAACAAGC AGAA GAGGGC GCCCUCU GCCGCUUGUUC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 17 AACGAACA AGAA GCAGAGCUCUGCC GCU UGUUCGUU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 108 GGAAGCGCAGAA GCCGCC GGCGGCG GCC GCGCUUCC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA120 GGAAGGGG AGAA GGAAGC GCUUCCG GCU CCCCUUCCACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 155 GUCGUUGA AGAA GAGCGC GCGCUCCGCC UCAACGAC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 176 CGGGGAGA AGAAGAGCGC GCGCUCU GCC UCUCCCCG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 186CGGCGAGC AGAA GGGAGA UCUCCCC GCC GCUCGCCGACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 189 GGGCGGCG AGAA GCGGGG CCCCGCCGCU CGCCGCCC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 196 CGGCGGCG AGAAGCGAGC GCUCGCC GCC CGCCGCCG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 200GCGGCGGC AGAA GGCGGC GCCGCCC GCC GCCGCCGCACCAGAGAAACACACGUUGUGGUACAUUACCUGCUA 203 GCGGCGGC AGAA GCGGGC GCCCGCCGCC GCCGCCGC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 206 GCUGCGGC AGAAGCGGCG CGCCGCC GCC GCCGCAGC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 209GCUGCUGC AGAA GCGGCG CGCCGCC GCC GCAGCAGCACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 235 AUGGAGGC AGAA GCGACG CGUCGCCGUC GCCUCCAU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 253 GUGGAGAC AGAAGACGUC GACGUCC GCC GUCUCCAC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 256UUGGUGGA AGAA GCGGAC GUCCGCC GUC UCCACCAAACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 406 CACUUCUC AGAA GGCUUG CAAGCCAGUC GAGAAGUG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 442 GAUGCUGG AGAAGGGAGG CCUCCCG GAC CCAGCAUC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGGA 508AAAUAAUC AGAA GGGAUU AAUCCCU GAU GAUUAUUUACCAGAGAAACACACGUUGUGGUACAUUACCUCGUA 570 UAAGCAUA AGAA GGUAUG CAUACCAGAC UAUGCUUA ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 625 ACAGCCCA AGAAGUGGGG CCCCACU GCC UGGGCUGU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 634CUCGUCCA AGAA GCCCAG CUGGGCU GUU UGGACGAGACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 655 UUCUCCUC AGAA GUCCAU AUGGACUGCU GAGGAGAA ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 681 ACUUGUUG AGAAGAUCAC GUGAUCU GCU CAACAAGU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 726UCUUCUCA AGAA GCCUCA UGAGGCA GAU UGAGAAGAACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 853 GCGUGACG AGAA GUGUUC GAACACUGCU CGUCACGC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 916 CGCUUCUC AGAAGAGGCG CGCCUCA GAU GAGAAGCG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 963CGAUCUCA AGAA GCUUCU AGAAGCU GUU UGAGAUCGACCAGAGAAACACACCUUGUCGUACAUUACCUGGUA 979 ACGGUACC AGAA GGGUCG CGACCCUGAU GGUACCGU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 1033 AUCAGGUG AGAAGGCAUU AAUGCCU GCC CACCUGAU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 1041CGUCAAAC AGAA GGUGGG CCCACCU GAU GUUUGACGACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 1068 AGUGCUCG AGAA GCUUGU ACAAGCUGUU CGAGCACU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 1173 ACAGACCA AGAAGGCUCG CGAGCCU GAC UGGUCUGU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 1182CUUCACCC AGAA GACCAG CUGGUCU GUC GGGUGAAGACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 1287 AGCUGAAA AGAA GCGUGC GCACGCUGCC UUUCAGCU ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 1295 GUAUACCC AGAAGAAAGG CCUUUCA GCU GGGUAUAC ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 1339CAGACCGC AGAA GGUUUC GAAACCU GCU GCGGUCUGACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 1345 UCUAAGCA AGAA GCAGCA UGCUGCGGUC UGCUUAGA ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 1349 CUUGUCUA AGAAGACCGC GCGGUCU GCU UAGACAAG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 1364GCAGACAC AGAA GGUCUU AAGACCU GCU GUGUCUGCACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 1483 UACAUCAA AGAA GAAAAA UUUUUCUGCU UUGAUGUA ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 1554 CCGCCAGC AGAAGACAAA UUUGUCU GUC GCUGGCGG ACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA 1595UUUAACAG AGAA GAGCAA UUGCUCA GAU CUGUUAAAACCAGAGAAACACACGUUGUGGUACAUUACCUGGUA

TABLE IX Cleavage of Delta-9 Desaturase RNA by HH Ribozymes PercentCleaved 20° C. 26° C. nt. Position 10 min 120 min 10 min 120 min 183 6.37.0 10.45 11.8 252 25.2 51.2 33.1 52.9 259 20.3 41.3 24.8 44.0 271 17.252.4 21.5 56.3 278 9.9 25.7 13.3 33.6 307 10.3 24.2 9.2 32.4 313 16.943.0 23.8 53.4 320 10.6 23.6 15.0 31.3 326 5.7 14.6 8.0 17.1 338 10.017.5 10.4 12.9 353 10.2 11.3 10.7 14.7 390 8.6 8.9 7.8 9.8 419 6.3 10.15.8 10.9 453 7.3 29.0 8.0 33.8 484 7.8 28.9 6.9 29.2 545 4.8 8.5 3.6 8.9773 4.5 11.5 4.4 8.9 1024 11.9 17.1 13.3 23.8 1026 11.6 12.6 13.1 17.21237 23.1 32.4 13.8 28.6

TABLE X Construct Targets Isolates Greenhouse Plants Number BlastedRecovered Lines Produced RPA85 231 70 13 161 RPA113 292 82 9 116 RPA114244 35 12 152 RPA115 285 42 11 165 RPA118 268 38 10 125 RPA119 301 67 11135 Totals 1621 334 66 854

TABLE XI Stearic acid levels in leaves from plants transformed withactive and inactive ribozymes compared to control leaves. Stearic Acidin Leaves Transformed with Active and Inactive Ribozymes (Percentage oftotal plants with certain levels of leaf stearic acid) Ribozyme RibozymeActives Inactives (428 plants (406 plants Controls Stearic Acid from 35lines) from 31 lines) (122 plants) >3% 7%    3%    2% >5% 2% 0 0 >10% 0    0 0

TABLE XII Inheritance of the high stearic acid trait in leaves fromcrosses of high stearic acid plants. Inheritance of high stearate inleaves. R1 Plants R1 Plants % of Plants with Normal with High with HighCross Leaf Stearate Leaf Stearate Stearate RPA85-15.06 × RPA85-15.12 6 333% RPA85-15.07 self 5 5 50% RPA85-15.10 self 8 2 20% OQ414 ×RPA85-15.06 5 3 38% OQ414 × RPA85-15.11 6 4 40%

TABLE XIII Comparison of fatty acid composition of embryogenic callus,somatic embryos and zygotic embryos. Tissue and/ % Lipid or Media FattyAcid Composition of Fresh Treatment C16:0 C18:0 C18:1 C18:2 C18:3 Weightembryogenic 19.4 1.1 6.2 55.7 8.8 0.4 callus +/− +/− +/− +/− +/− +/− 0.90.1 2.0 3.1 2.0 0.1 somatic 12.6 1.6 18.2 60.7 1.9 4.0 embryo grown +/−+/− +/− +/− +/− +/− on MS + 6% 0.7 0.8 4.9 5.1 0.3 1.1 sucrose + 10 mMABA zygotic embryo 14.5 1.1 18.5 60.2 1.4 3.9 12 days after +/− +/− +/−+/− +/− +/− pollination 0.4 0.1 1.0 1.5 0.2 0.6

TABLE XIV GBSS activity, amylose content, and Southern analysis resultsof selected Ribozyme Lines GBSS activity Amylose Content Line (Units/mgstarch) (%) Southern RPA63.0283 321.5 ± 31.2 23.3 ± 0.5 − RPA63.0236314.6 ± 9.2  27.4 ± 0.3 − RPA63.0219 299.8 ± 10.4 21.5 ± 0.3 −RPA63.0314 440.4 ± 17.1 19.1 ± 0.8 − RPA63.0316 346.5 ± 8.5  17.9 ± 0.5− RPA63.0311 301.5 ± 17.4 19.5 ± 0.4 − RPA63.0309 264.7 ± 19   21.7 ±0.1 + RPA63.0218 190.8 ± 7.8  21.0 ± 0.3 + RPA63.0209  203 ± 2.4 22.6 ±0.6 + RPA63.0306 368.2 ± 7.5  19.0 ± 0.4 − RPA63.0210 195.1 ± 7   22.1 ±0.2 +

1263 1621 base pairs nucleic acid single linear Coding Sequence146...1324 1 CGCACGCGCC CTCTGCCGCT TGTTCGTTCC TCGCGCTCGC CACCAGGCACCACCACACAC 60 ATCCCAATCT CGCGAGGGCA AGCAGCAGGG TCTGCGGCGG CGGCGGCGGCCGCGCTTCCG 120 GCTCCCCTTC CCATTGGCCT CCACG ATG GCG CTC CGC CTC AAC GACGTC GCG 172 Met Ala Leu Arg Leu Asn Asp Val Ala 1 5 CTC TGC CTC TCC CCGCCG CTC GCC GCC CGC CGC CGC CGC CGC AGC AGC 220 Leu Cys Leu Ser Pro ProLeu Ala Ala Arg Arg Arg Arg Arg Ser Ser 10 15 20 25 GGC AGG TTC GTC GCCGTC GCC TCC ATG ACG TCC GCC GTC TCC ACC AAG 268 Gly Arg Phe Val Ala ValAla Ser Met Thr Ser Ala Val Ser Thr Lys 30 35 40 GTC GAG AAT AAG AAG CCATTT GCT CCT CCA AGG GAG GTA CAT GTC CAG 316 Val Glu Asn Lys Lys Pro PheAla Pro Pro Arg Glu Val His Val Gln 45 50 55 GTT ACA CAT TCA ATG CCA CCTCAC AAG ATT GAA ATT TTC AAG TCG CTT 364 Val Thr His Ser Met Pro Pro HisLys Ile Glu Ile Phe Lys Ser Leu 60 65 70 GAT GAT TGG GCT AGA GAT AAT ATCTTG ACG CAT CTC AAG CCA GTC GAG 412 Asp Asp Trp Ala Arg Asp Asn Ile LeuThr His Leu Lys Pro Val Glu 75 80 85 AAG TGT TGG CAG CCA CAG GAT TTC CTCCCG GAC CCA GCA TCT GAA GGA 460 Lys Cys Trp Gln Pro Gln Asp Phe Leu ProAsp Pro Ala Ser Glu Gly 90 95 100 105 TTT CAT GAT GAA GTT AAG GAG CTCAGA GAA CGT GCC AAG GAA ATC CCT 508 Phe His Asp Glu Val Lys Glu Leu ArgGlu Arg Ala Lys Glu Ile Pro 110 115 120 GAT GAT TAT TTT GTT TGT TTG GTGGGA GAC ATG ATT ACC GAG GAA GCT 556 Asp Asp Tyr Phe Val Cys Leu Val GlyAsp Met Ile Thr Glu Glu Ala 125 130 135 CTA CCA ACA TAC CAG ACT ATG CTTAAC ACC CTC GAC GGT GTC AGA GAT 604 Leu Pro Thr Tyr Gln Thr Met Leu AsnThr Leu Asp Gly Val Arg Asp 140 145 150 GAG ACA GGT GCA AGC CCC ACT GCCTGG GCT GTT TGG ACG AGG GCA TGG 652 Glu Thr Gly Ala Ser Pro Thr Ala TrpAla Val Trp Thr Arg Ala Trp 155 160 165 ACT GCT GAG GAG AAC AGG CAT GGTGAT CTG CTC AAC AAG TAT ATG TAC 700 Thr Ala Glu Glu Asn Arg His Gly AspLeu Leu Asn Lys Tyr Met Tyr 170 175 180 185 CTC ACT GGG AGG GTG GAT ATGAGG CAG ATT GAG AAG ACA ATT CAG TAT 748 Leu Thr Gly Arg Val Asp Met ArgGln Ile Glu Lys Thr Ile Gln Tyr 190 195 200 CTT ATT GGC TCT GGA ATG GATCCT AGG ACT GAG AAT AAT CCT TAT CTT 796 Leu Ile Gly Ser Gly Met Asp ProArg Thr Glu Asn Asn Pro Tyr Leu 205 210 215 GGT TTC ATC TAC ACC TCC TTCCAA GAG CGG GCG ACC TTC ATC TCA CAC 844 Gly Phe Ile Tyr Thr Ser Phe GlnGlu Arg Ala Thr Phe Ile Ser His 220 225 230 GGG AAC ACT GCT CGT CAC GCCAAG GAC TTT GGC GAC TTA AAG CTT GCA 892 Gly Asn Thr Ala Arg His Ala LysAsp Phe Gly Asp Leu Lys Leu Ala 235 240 245 CAA ATC TGC GGC ATC ATC GCCTCA GAT GAG AAG CGA CAT GAA ACT GCG 940 Gln Ile Cys Gly Ile Ile Ala SerAsp Glu Lys Arg His Glu Thr Ala 250 255 260 265 TAC ACC AAG ATC GTG GAGAAG CTG TTT GAG ATC GAC CCT GAT GGT ACC 988 Tyr Thr Lys Ile Val Glu LysLeu Phe Glu Ile Asp Pro Asp Gly Thr 270 275 280 GTG GTC GCT CTG GCT GACATG ATG AGG AAG AAG ATC TCA ATG CCT GCC 1036 Val Val Ala Leu Ala Asp MetMet Arg Lys Lys Ile Ser Met Pro Ala 285 290 295 CAC CTG ATG TTT GAC GGGCAG GAC GAC AAG CTG TTC GAG CAC TTC TCC 1084 His Leu Met Phe Asp Gly GlnAsp Asp Lys Leu Phe Glu His Phe Ser 300 305 310 ATG GTC GCG CAG AGG CTTGGC GTT TAC ACC GCC AGG GAC TAC GCC GAC 1132 Met Val Ala Gln Arg Leu GlyVal Tyr Thr Ala Arg Asp Tyr Ala Asp 315 320 325 ATC CTC GAG TTC CTC GTCGAC AGG TGG AAG GTG GCG AGC CTG ACT GGT 1180 Ile Leu Glu Phe Leu Val AspArg Trp Lys Val Ala Ser Leu Thr Gly 330 335 340 345 CTG TCG GGT GAA GGGAAC AAG GCG CAG GAC TAC CTT TGC ACC CTT GCT 1228 Leu Ser Gly Glu Gly AsnLys Ala Gln Asp Tyr Leu Cys Thr Leu Ala 350 355 360 TCA AGA ATC AGG AGGCTG GAG GAG AGG GCC CAG AGC AGA GCC AAG AAA 1276 Ser Arg Ile Arg Arg LeuGlu Glu Arg Ala Gln Ser Arg Ala Lys Lys 365 370 375 GCC GGC ACG CTG CCTTTC AGC TGG GTA TAC GGT AGG GAC GTC CAA CTG TG 1326 Ala Gly Thr Leu ProPhe Ser Trp Val Tyr Gly Arg Asp Val Gln Leu 380 385 390 AGATCGGAAACCTGCTGCGG TCTGCTTAGA CAAGACCTGC TGTGTCTGCG TTACATAGGT 1386 CTCCAGGTTTTGATCAAATG GTCCCGTGTC GTCTTATAGA GCGATAGGAG AACGTGTTGG 1446 TCTGTGGTGTAGCTTTGTTT TTATTTTGTA TTTTTCTGCT TTGATGTACA ACCTGTGGCC 1506 GCATGAACTGGGGCGTGGAG ATGGGAGCGA CCATGCCGTA CTTTGTCTGT CGCTGGCGGT 1566 GTGTTTCGGTATGTTATTTG AGTTGCTCAG ATCTGTTAAA AAAAAAAAAA AAAAA 1621 42 base pairsnucleic acid single linear 2 CGACGAAGAC CUGAUGAGGC CGAAAGGCCG AAACGUUCAUGC 42 42 base pairs nucleic acid single linear 3 CUCCCAUCUU CUGAUGAGGCCGAAAGGCCG AAAUCUCGGA CA 42 42 base pairs nucleic acid single linear 4GUUGUCCCUG CUGAUGAGGC CGAAAGGCCG AAAGUCCGUU CC 42 42 base pairs nucleicacid single linear 5 GGUUGUUGUU CUGAUGAGGC CGAAAGGCCG AAAGGCUCAG GA 4242 base pairs nucleic acid single linear 6 GAGGUAGCAC CUGAUGAGGCCGAAAGGCCG AAAGAGAGGG CC 42 42 base pairs nucleic acid single linear 7GUGGGACUGG CUGAUGAGGC CGAAAGGCCG AAAGUUGCUC UU 42 42 base pairs nucleicacid single linear 8 GAUGCCGUGG CUGAUGAGGC CGAAAGGCCG AAACUGGUAG UU 4242 base pairs nucleic acid single linear 9 UGUGGAUGCA CUGAUGAGGCCGAAAGGCCG AAAAAGCGGU CU 42 42 base pairs nucleic acid single linear 10AGAUGUUGUG CUGAUGAGGC CGAAAGGCCG AAAUGCAGAA AG 42 42 base pairs nucleicacid single linear 11 CCUGGUAGGA CUGAUGAGGC CGAAAGGCCG AAAUGUUGUG GA 4242 base pairs nucleic acid single linear 12 AUCUCUCCGG CUGAUGAGGCCGAAAGGCCG AAAGGUUCAG CU 42 42 base pairs nucleic acid single linear 13AGGACGACUU CUGAUGAGGC CGAAAGGCCG AAAAUCUCUC CG 42 42 base pairs nucleicacid single linear 14 UCAUCCAGUU CUGAUGAGGC CGAAAGGCCG AAAUCUUCCG GC 4242 base pairs nucleic acid single linear 15 UGAUGUUGUC CUGAUGAGGCCGAAAGGCCG AAAGCUCGCA GC 42 42 base pairs nucleic acid single linear 16UGAGGCGCAU CUGAUGAGGC CGAAAGGCCG AAAUGUUGUC GA 42 42 base pairs nucleicacid single linear 17 CCAUGCCGUU CUGAUGAGGC CGAAAGGCCG AAACGAUGCC GG 4242 base pairs nucleic acid single linear 18 CCCACUCGCU CUGAUGAGGCCGAAAGGCCG AAACGUCCAU GC 42 42 base pairs nucleic acid single linear 19CACGGCGAUG CUGAUGAGGC CGAAAGGCCG AAACUUGUCC CU 42 42 base pairs nucleicacid single linear 20 GGUCCACCGG CUGAUGAGGC CGAAAGGCCG AAAGCCCGAC CU 4242 base pairs nucleic acid single linear 21 CGGCCGCCAU CUGAUGAGGCCGAAAGGCCG AAACGUCGGG UC 42 42 base pairs nucleic acid single linear 22CUUGCCUGGG CUGAUGAGGC CGAAAGGCCG AAACUUCUCC UC 42 42 base pairs nucleicacid single linear 23 UGCCUUCGAU CUGAUGAGGC CGAAAGGCCG AAAUGGUGUC GA 4242 base pairs nucleic acid single linear 24 CCACCUUCUU CUGAUGAGGCCGAAAGGCCG AAACGUCCGC CG 42 2267 base pairs nucleic acid single linear25 GACCGATCGA TCGCCACAGC CAACACCACC CGCCGAGGCG ACGCGACAGC CGCCAGGAGG 60AAGGAATAAA CTCACTGCCA GCCAGTGAAG GGGGAGAAGT GTACTGCTCC GTCCACCAGT 120GCGCGCACCG CCCGGCAGGG CTGCTCATCT CGTCGACGAC CAGTGGATTA ATCGGCATGG 180CGGCTCTAGC CACGTCGCAG CTCGTCGCAA CGCGCGCCGG CCTGGGCGTC CCGGACGCGT 240CCACGTTCCG CCGCGGCGCC GCGCAGGGCC TGAGGGGGGG CCGGACGGCG TCGGCGGCGG 300ACACGCTCAG CATTCGGACC AGCGCGCGCG CGGCGCCCAG GCTCCAGCAC CAGCAGCAGC 360AGCAGGCGCG CCGCGGGGCC AGGTTCCCGT CGCTCGTCGT GTGCGCCAGC GCCGGCATGA 420ACGTCGTCTT CGTCGGCGCC GAGATGGCGC CGTGGAGCAA GACCGGCGGC CTCGGCGACG 480TCCTCGGCGG CCTGCCGCCG GCCATGGCCG CGAATGGGCA CCGTGTCATG GTCGTCTCTC 540CCCGCTACGA CCAGTACAAG GACGCCTGGG ACACCAGCGT CGTGTCCGAG ATCAAGATGG 600GAGACAGGTA CGAGACGGTC AGGTTCTTCC ACTGCTACAA GCGCGGAGTG GACCGCGTGT 660TCGTTGACCA CCCACTGTTC CTGGAGAGGG TTTGGGGAAA GACCGAGGAG AAGATCTACG 720GGCCTGACGC TGGAACGGAC TACAGGGACA ACCAGCTGCG GTTCAGCCTG CTATGCCAGG 780CAGCACTTGA AGCTCCAAGG ATCCTGAGCC TCAACAACAA CCCATACTTC TCCGGACCAT 840ACGGGGAGGA CGTCGTGTTC GTCTGCAACG ACTGGCACAC CGGCCCTCTC TCGTGCTACC 900TCAAGAGCAA CTACCAGTCC CACGGCATCT ACAGGGACGC AAAGACCGCT TTCTGCATCC 960ACAACATCTC CTACCAGGGC CGGTTCGCCT TCTCCGACTA CCCGGAGCTG AACCTCCCGG 1020AGAGATTCAA GTCGTCCTTC GATTTCATCG ACGGCTACGA GAAGCCCGTG GAAGGCCGGA 1080AGATCAACTG GATGAAGGCC GGGATCCTCG AGGCCGACAG GGTCCTCACC GTCAGCCCCT 1140ACTACGCCGA GGAGCTCATC TCCGGCATCG CCAGGGGCTG CGAGCTCGAC AACATCATGC 1200GCCTCACCGG CATCACCGGC ATCGTCAACG GCATGGACGT CAGCGAGTGG GACCCCAGCA 1260GGGACAAGTA CATCGCCGTG AAGTACGACG TGTCGACGGC CGTGGAGGCC AAGGCGCTGA 1320ACAAGGAGGC GCTGCAGGCG GAGGTCGGGC TCCCGGTGGA CCGGAACATC CCGCTGGTGG 1380CGTTCATCGG CAGGCTGGAA GAGCAGAAGG GACCCGACGT CATGGCGGCC GCCATCCCGC 1440AGCTCATGGA GATGGTGGAG GACGTGCAGA TCGTTCTGCT GGGCACGGGC AAGAAGAAGT 1500TCGAGCGCAT GCTCATGAGC GCCGAGGAGA AGTTCCCAGG CAAGGTGCGC GCCGTGGTCA 1560AGTTCAACGC GGCGCTGGCG CACCACATCA TGGCCGGCGC CGACGTGCTC GCCGTCACCA 1620GCCGCTTCGA GCCCTGCGGC CTCATCCAGC TGCAGGGGAT GCGATACGGA ACGCCCTGCG 1680CCTGCGCGTC CACCGGTGGA CTCGTCGACA CCATCATCGA AGGCAAGACC GGGTTCCACA 1740TGGGCCGCCT CAGCGTCGAC TGCAACGTCG TGGAGCCGGC GGACGTCAAG AAGGTGGCCA 1800CCACCTTGCA GCGCGCCATC AAGGTGGTCG GCACGCCGGC GTACGAGGAG ATGGTGAGGA 1860ACTGCATGAT CCAGGATCTC TCCTGGAAGG GCCCTGCCAA GAACTGGGAG AACGTGCTGC 1920TCAGCCTCGG GGTCGCCGGC GGCGAGCCAG GGGTCGAAGG CGAGGAGATC GCGCCGCTCG 1980CCAAGGAGAA CGTGGCCGCG CCCTGAAGAG TTCGGCCTGC AGGCCCCCTG ATCTCGCGCG 2040TGGTGCAAAC ATGTTGGGAC ATCTTCTTAT ATATGCTGTT TCGTTTATGT GATATGGACA 2100AGTATGTGTA GCTGCTTGCT TGTGCTAGTG TAATATAGTG TAGTGGTGGC CAGTGGCACA 2160ACCTAATAAG CGCATGAACT AATTGCTTGC GTGTGTAGTT AAGTACCGAT CGGTAATTTT 2220ATATTGCGAG TAAATAAATG GACCTGTAGT GGTGGAAAAA AAAAAAA 2267 17 base pairsnucleic acid single linear 26 CGAUCGAUCG CCACAGC 17 17 base pairsnucleic acid single linear 27 GGUCGUCUCU CCCCGCU 17 17 base pairsnucleic acid single linear 28 GAAGGAAUAA ACUCACU 17 17 base pairsnucleic acid single linear 29 UCGUCUCUCC CCGCUAC 17 17 base pairsnucleic acid single linear 30 AAUAAACUCA CUGCCAG 17 17 base pairsnucleic acid single linear 31 UCCCCGCUAC GACCAGU 17 17 base pairsnucleic acid single linear 32 AGAAGUGUAC UGCUCCG 17 17 base pairsnucleic acid single linear 33 CGACCAGUAC AAGGACG 17 17 base pairsnucleic acid single linear 34 GUACUGCUCC GUCCACC 17 17 base pairsnucleic acid single linear 35 ACCAGCGUCG UGUCCGA 17 17 base pairsnucleic acid single linear 36 UGCUCCGUCC ACCAGUG 17 17 base pairsnucleic acid single linear 37 CGUCGUGUCC GAGAUCA 17 17 base pairsnucleic acid single linear 38 GGGCUGCUCA UCUCGUC 17 17 base pairsnucleic acid single linear 39 UCCGAGAUCA AGAUGGG 17 17 base pairsnucleic acid single linear 40 CUGCUCAUCU CGUCGAC 17 17 base pairsnucleic acid single linear 41 AGACAGGUAC GAGACGG 17 17 base pairsnucleic acid single linear 42 GCUCAUCUCG UCGACGA 17 17 base pairsnucleic acid single linear 43 GAGACGGUCA GGUUCUU 17 17 base pairsnucleic acid single linear 44 CAUCUCGUCG ACGACCA 17 17 base pairsnucleic acid single linear 45 GGUCAGGUUC UUCCACU 17 17 base pairsnucleic acid single linear 46 CAGUGGAUUA AUCGGCA 17 17 base pairsnucleic acid single linear 47 GUCAGGUUCU UCCACUG 17 17 base pairsnucleic acid single linear 48 AGUGGAUUAA UCGGCAU 17 17 base pairsnucleic acid single linear 49 CAGGUUCUUC CACUGCU 17 17 base pairsnucleic acid single linear 50 GGAUUAAUCG GCAUGGC 17 17 base pairsnucleic acid single linear 51 AGGUUCUUCC ACUGCUA 17 17 base pairsnucleic acid single linear 52 UGGCGGCUCU AGCCACG 17 17 base pairsnucleic acid single linear 53 CCACUGCUAC AAGCGCG 17 17 base pairsnucleic acid single linear 54 GCGGCUCUAG CCACGUC 17 17 base pairsnucleic acid single linear 55 CCGCGUGUUC GUUGACC 17 17 base pairsnucleic acid single linear 56 AGCCACGUCG CAGCUCG 17 17 base pairsnucleic acid single linear 57 CGCGUGUUCG UUGACCA 17 17 base pairsnucleic acid single linear 58 UCGCAGCUCG UCGCAAC 17 17 base pairsnucleic acid single linear 59 GUGUUCGUUG ACCACCC 17 17 base pairsnucleic acid single linear 60 CAGCUCGUCG CAACGCG 17 17 base pairsnucleic acid single linear 61 CCCACUGUUC CUGGAGA 17 17 base pairsnucleic acid single linear 62 CUGGGCGUCC CGGACGC 17 17 base pairsnucleic acid single linear 63 CCACUGUUCC UGGAGAG 17 17 base pairsnucleic acid single linear 64 GGACGCGUCC ACGUUCC 17 17 base pairsnucleic acid single linear 65 GAGAGGGUUU GGGGAAA 17 17 base pairsnucleic acid single linear 66 GUCCACGUUC CGCCGCG 17 17 base pairsnucleic acid single linear 67 AGAGGGUUUG GGGAAAG 17 17 base pairsnucleic acid single linear 68 UCCACGUUCC GCCGCGG 17 17 base pairsnucleic acid single linear 69 GAGAAGAUCU ACGGGCC 17 17 base pairsnucleic acid single linear 70 GACGGCGUCG GCGGCGG 17 17 base pairsnucleic acid single linear 71 GAAGAUCUAC GGGCCUG 17 17 base pairsnucleic acid single linear 72 GACACGCUCA GCAUUCG 17 17 base pairsnucleic acid single linear 73 AACGGACUAC AGGGACA 17 17 base pairsnucleic acid single linear 74 CUCAGCAUUC GGACCAG 17 17 base pairsnucleic acid single linear 75 GCUGCGGUUC AGCCUGC 17 17 base pairsnucleic acid single linear 76 UCAGCAUUCG GACCAGC 17 17 base pairsnucleic acid single linear 77 CUGCGGUUCA GCCUGCU 17 17 base pairsnucleic acid single linear 78 CCCAGGCUCC AGCACCA 17 17 base pairsnucleic acid single linear 79 AGCCUGCUAU GCCAGGC 17 17 base pairsnucleic acid single linear 80 GGCCAGGUUC CCGUCGC 17 17 base pairsnucleic acid single linear 81 GCAGCACUUG AAGCUCC 17 17 base pairsnucleic acid single linear 82 GCCAGGUUCC CGUCGCU 17 17 base pairsnucleic acid single linear 83 UUGAAGCUCC AAGGAUC 17 17 base pairsnucleic acid single linear 84 GUUCCCGUCG CUCGUCG 17 17 base pairsnucleic acid single linear 85 CCAAGGAUCC UGAGCCU 17 17 base pairsnucleic acid single linear 86 CCGUCGCUCG UCGUGUG 17 17 base pairsnucleic acid single linear 87 CUGAGCCUCA ACAACAA 17 17 base pairsnucleic acid single linear 88 UCGCUCGUCG UGUGCGC 17 17 base pairsnucleic acid single linear 89 CAACCCAUAC UUCUCCG 17 17 base pairsnucleic acid single linear 90 AUGAACGUCG UCUUCGU 17 17 base pairsnucleic acid single linear 91 CCCAUACUUC UCCGGAC 17 17 base pairsnucleic acid single linear 92 AACGUCGUCU UCGUCGG 17 17 base pairsnucleic acid single linear 93 CCAUACUUCU CCGGACC 17 17 base pairsnucleic acid single linear 94 CGUCGUCUUC GUCGGCG 17 17 base pairsnucleic acid single linear 95 AUACUUCUCC GGACCAU 17 17 base pairsnucleic acid single linear 96 GUCGUCUUCG UCGGCGC 17 17 base pairsnucleic acid single linear 97 CGGACCAUAC GGGGAGG 17 17 base pairsnucleic acid single linear 98 GUCUUCGUCG GCGCCGA 17 17 base pairsnucleic acid single linear 99 GAGGACGUCG UGUUCGU 17 17 base pairsnucleic acid single linear 100 GGCGGCCUCG GCGACGU 17 17 base pairsnucleic acid single linear 101 CGUCGUGUUC GUCUGCA 17 17 base pairsnucleic acid single linear 102 GGCGACGUCC UCGGCGG 17 17 base pairsnucleic acid single linear 103 GUCGUGUUCG UCUGCAA 17 17 base pairsnucleic acid single linear 104 GACGUCCUCG GCGGCCU 17 17 base pairsnucleic acid single linear 105 GUGUUCGUCU GCAACGA 17 17 base pairsnucleic acid single linear 106 CACCGUGUCA UGGUCGU 17 17 base pairsnucleic acid single linear 107 CCGGCCCUCU CUCGUGC 17 17 base pairsnucleic acid single linear 108 GUCAUGGUCG UCUCUCC 17 17 base pairsnucleic acid single linear 109 GGCCCUCUCU CGUGCUA 17 17 base pairsnucleic acid single linear 110 AUGGUCGUCU CUCCCCG 17 17 base pairsnucleic acid single linear 111 CCCUCUCUCG UGCUACC 17 17 base pairsnucleic acid single linear 112 CUCGUGCUAC CUCAAGA 17 17 base pairsnucleic acid single linear 113 AUGGACGUCA GCGAGUG 17 17 base pairsnucleic acid single linear 114 UGCUACCUCA AGAGCAA 17 17 base pairsnucleic acid single linear 115 GGACAAGUAC AUCGCCG 17 17 base pairsnucleic acid single linear 116 GAGCAACUAC CAGUCCC 17 17 base pairsnucleic acid single linear 117 AAGUACAUCG CCGUGAA 17 17 base pairsnucleic acid single linear 118 CUACCAGUCC CACGGCA 17 17 base pairsnucleic acid single linear 119 CGUGAAGUAC GACGUGU 17 17 base pairsnucleic acid single linear 120 CACGGCAUCU ACAGGGA 17 17 base pairsnucleic acid single linear 121 CGACGUGUCG ACGGCCG 17 17 base pairsnucleic acid single linear 122 CGGCAUCUAC AGGGACG 17 17 base pairsnucleic acid single linear 123 GCGGAGGUCG GGCUCCC 17 17 base pairsnucleic acid single linear 124 AGACCGCUUU CUGCAUC 17 17 base pairsnucleic acid single linear 125 GUCGGGCUCC CGGUGGA 17 17 base pairsnucleic acid single linear 126 GACCGCUUUC UGCAUCC 17 17 base pairsnucleic acid single linear 127 CGGAACAUCC CGCUGGU 17 17 base pairsnucleic acid single linear 128 ACCGCUUUCU GCAUCCA 17 17 base pairsnucleic acid single linear 129 GGUGGCGUUC AUCGGCA 17 17 base pairsnucleic acid single linear 130 UUCUGCAUCC ACAACAU 17 17 base pairsnucleic acid single linear 131 GUGGCGUUCA UCGGCAG 17 17 base pairsnucleic acid single linear 132 CACAACAUCU CCUACCA 17 17 base pairsnucleic acid single linear 133 GCGUUCAUCG GCAGGCU 17 17 base pairsnucleic acid single linear 134 CAACAUCUCC UACCAGG 17 17 base pairsnucleic acid single linear 135 CCCGACGUCA UGGCGGC 17 17 base pairsnucleic acid single linear 136 CAUCUCCUAC CAGGGCC 17 17 base pairsnucleic acid single linear 137 GCCGCCAUCC CGCAGCU 17 17 base pairsnucleic acid single linear 138 GGGCCGGUUC GCCUUCU 17 17 base pairsnucleic acid single linear 139 CCGCAGCUCA UGGAGAU 17 17 base pairsnucleic acid single linear 140 GGCCGGUUCG CCUUCUC 17 17 base pairsnucleic acid single linear 141 GUGCAGAUCG UUCUGCU 17 17 base pairsnucleic acid single linear 142 GUUCGCCUUC UCCGACU 17 17 base pairsnucleic acid single linear 143 CAGAUCGUUC UGCUGGG 17 17 base pairsnucleic acid single linear 144 UUCGCCUUCU CCGACUA 17 17 base pairsnucleic acid single linear 145 AGAUCGUUCU GCUGGGC 17 17 base pairsnucleic acid single linear 146 CGCCUUCUCC GACUACC 17 17 base pairsnucleic acid single linear 147 GAAGAAGUUC GAGCGCA 17 17 base pairsnucleic acid single linear 148 CUCCGACUAC CCGGAGC 17 17 base pairsnucleic acid single linear 149 AAGAAGUUCG AGCGCAU 17 17 base pairsnucleic acid single linear 150 CUGAACCUCC CGGAGAG 17 17 base pairsnucleic acid single linear 151 CGCAUGCUCA UGAGCGC 17 17 base pairsnucleic acid single linear 152 GGAGAGAUUC AAGUCGU 17 17 base pairsnucleic acid single linear 153 GGAGAAGUUC CCAGGCA 17 17 base pairsnucleic acid single linear 154 GAGAGAUUCA AGUCGUC 17 17 base pairsnucleic acid single linear 155 GAGAAGUUCC CAGGCAA 17 17 base pairsnucleic acid single linear 156 AUUCAAGUCG UCCUUCG 17 17 base pairsnucleic acid single linear 157 GCCGUGGUCA AGUUCAA 17 17 base pairsnucleic acid single linear 158 CAAGUCGUCC UUCGAUU 17 17 base pairsnucleic acid single linear 159 GGUCAAGUUC AACGCGG 17 17 base pairsnucleic acid single linear 160 GUCGUCCUUC GAUUUCA 17 17 base pairsnucleic acid single linear 161 GUCAAGUUCA ACGCGGC 17 17 base pairsnucleic acid single linear 162 UCGUCCUUCG AUUUCAU 17 17 base pairsnucleic acid single linear 163 CACCACAUCA UGGCCGG 17 17 base pairsnucleic acid single linear 164 CCUUCGAUUU CAUCGAC 17 17 base pairsnucleic acid single linear 165 GACGUGCUCG CCGUCAC 17 17 base pairsnucleic acid single linear 166 CUUCGAUUUC AUCGACG 17 17 base pairsnucleic acid single linear 167 CUCGCCGUCA CCAGCCG 17 17 base pairsnucleic acid single linear 168 UUCGAUUUCA UCGACGG 17 17 base pairsnucleic acid single linear 169 CAGCCGCUUC GAGCCCU 17 17 base pairsnucleic acid single linear 170 GAUUUCAUCG ACGGCUA 17 17 base pairsnucleic acid single linear 171 AGCCGCUUCG AGCCCUG 17 17 base pairsnucleic acid single linear 172 CGACGGCUAC GAGAAGC 17 17 base pairsnucleic acid single linear 173 UGCGGCCUCA UCCAGCU 17 17 base pairsnucleic acid single linear 174 CGGAAGAUCA ACUGGAU 17 17 base pairsnucleic acid single linear 175 GGCCUCAUCC AGCUGCA 17 17 base pairsnucleic acid single linear 176 GCCGGGAUCC UCGAGGC 17 17 base pairsnucleic acid single linear 177 GAUGCGAUAC GGAACGC 17 17 base pairsnucleic acid single linear 178 GGGAUCCUCG AGGCCGA 17 17 base pairsnucleic acid single linear 179 CUGCGCGUCC ACCGGUG 17 17 base pairsnucleic acid single linear 180 GACAGGGUCC UCACCGU 17 17 base pairsnucleic acid single linear 181 GGUGGACUCG UCGACAC 17 17 base pairsnucleic acid single linear 182 AGGGUCCUCA CCGUCAG 17 17 base pairsnucleic acid single linear 183 GGACUCGUCG ACACCAU 17 17 base pairsnucleic acid single linear 184 CUCACCGUCA GCCCCUA 17 17 base pairsnucleic acid single linear 185 GACACCAUCA UCGAAGG 17 17 base pairsnucleic acid single linear 186 CAGCCCCUAC UACGCCG 17 17 base pairsnucleic acid single linear 187 ACCAUCAUCG AAGGCAA 17 17 base pairsnucleic acid single linear 188 CCCCUACUAC GCCGAGG 17 17 base pairsnucleic acid single linear 189 GACCGGGUUC CACAUGG 17 17 base pairsnucleic acid single linear 190 GAGGAGCUCA UCUCCGG 17 17 base pairsnucleic acid single linear 191 ACCGGGUUCC ACAUGGG 17 17 base pairsnucleic acid single linear 192 GAGCUCAUCU CCGGCAU 17 17 base pairsnucleic acid single linear 193 GGCCGCCUCA GCGUCGA 17 17 base pairsnucleic acid single linear 194 GCUCAUCUCC GGCAUCG 17 17 base pairsnucleic acid single linear 195 CUCAGCGUCG ACUGCAA 17 17 base pairsnucleic acid single linear 196 UCCGGCAUCG CCAGGGG 17 17 base pairsnucleic acid single linear 197 UGCAACGUCG UGGAGCC 17 17 base pairsnucleic acid single linear 198 UGCGAGCUCG ACAACAU 17 17 base pairsnucleic acid single linear 199 GCGGACGUCA AGAAGGU 17 17 base pairsnucleic acid single linear 200 GACAACAUCA UGCGCCU 17 17 base pairsnucleic acid single linear 201 CACCACCUUG CAGCGCG 17 17 base pairsnucleic acid single linear 202 AUGCGCCUCA CCGGCAU 17 17 base pairsnucleic acid single linear 203 CGCGCCAUCA AGGUGGU 17 17 base pairsnucleic acid single linear 204 ACCGGCAUCA CCGGCAU 17 17 base pairsnucleic acid single linear 205 AAGGUGGUCG GCACGCC 17 17 base pairsnucleic acid single linear 206 ACCGGCAUCG UCAACGG 17 17 base pairsnucleic acid single linear 207 GCCGGCGUAC GAGGAGA 17 17 base pairsnucleic acid single linear 208 GGCAUCGUCA ACGGCAU 17 17 base pairsnucleic acid single linear 209 UGCAUGAUCC AGGAUCU 17 17 base pairsnucleic acid single linear 210 UCCAGGAUCU CUCCUGG 17 17 base pairsnucleic acid single linear 211 CGGUAAUUUU AUAUUGC 17 17 base pairsnucleic acid single linear 212 CAGGAUCUCU CCUGGAA 17 17 base pairsnucleic acid single linear 213 GGUAAUUUUA UAUUGCG 17 17 base pairsnucleic acid single linear 214 GGAUCUCUCC UGGAAGG 17 17 base pairsnucleic acid single linear 215 GUAAUUUUAU AUUGCGA 17 17 base pairsnucleic acid single linear 216 GUGCUGCUCA GCCUCGG 17 17 base pairsnucleic acid single linear 217 AAUUUUAUAU UGCGAGU 17 17 base pairsnucleic acid single linear 218 CUCAGCCUCG GGGUCGC 17 17 base pairsnucleic acid single linear 219 UUUUAUAUUG CGAGUAA 17 17 base pairsnucleic acid single linear 220 CUCGGGGUCG CCGGCGG 17 17 base pairsnucleic acid single linear 221 UUGCGAGUAA AUAAAUG 17 17 base pairsnucleic acid single linear 222 CCAGGGGUCG AAGGCGA 17 17 base pairsnucleic acid single linear 223 GAGUAAAUAA AUGGACC 17 17 base pairsnucleic acid single linear 224 GAGGAGAUCG CGCCGCU 17 17 base pairsnucleic acid single linear 225 GGACCUGUAG UGGUGGA 17 17 base pairsnucleic acid single linear 226 GCGCCGCUCG CCAAGGA 17 17 base pairsnucleic acid single linear 227 UGAAGAGUUC GGCCUGC 17 17 base pairsnucleic acid single linear 228 GAAGAGUUCG GCCUGCA 17 17 base pairsnucleic acid single linear 229 CCCCUGAUCU CGCGCGU 17 17 base pairsnucleic acid single linear 230 CCUGAUCUCG CGCGUGG 17 17 base pairsnucleic acid single linear 231 AAACAUGUUG GGACAUC 17 17 base pairsnucleic acid single linear 232 UGGGACAUCU UCUUAUA 17 17 base pairsnucleic acid single linear 233 GGACAUCUUC UUAUAUA 17 17 base pairsnucleic acid single linear 234 GACAUCUUCU UAUAUAU 17 17 base pairsnucleic acid single linear 235 CAUCUUCUUA UAUAUGC 17 17 base pairsnucleic acid single linear 236 AUCUUCUUAU AUAUGCU 17 17 base pairsnucleic acid single linear 237 CUUCUUAUAU AUGCUGU 17 17 base pairsnucleic acid single linear 238 UCUUAUAUAU GCUGUUU 17 17 base pairsnucleic acid single linear 239 UAUGCUGUUU CGUUUAU 17 17 base pairsnucleic acid single linear 240 AUGCUGUUUC GUUUAUG 17 17 base pairsnucleic acid single linear 241 UGCUGUUUCG UUUAUGU 17 17 base pairsnucleic acid single linear 242 UGUUUCGUUU AUGUGAU 17 17 base pairsnucleic acid single linear 243 GUUUCGUUUA UGUGAUA 17 17 base pairsnucleic acid single linear 244 UUUCGUUUAU GUGAUAU 17 17 base pairsnucleic acid single linear 245 UAUGUGAUAU GGACAAG 17 17 base pairsnucleic acid single linear 246 GGACAAGUAU GUGUAGC 17 17 base pairsnucleic acid single linear 247 GUAUGUGUAG CUGCUUG 17 17 base pairsnucleic acid single linear 248 UAGCUGCUUG CUUGUGC 17 17 base pairsnucleic acid single linear 249 UGCUUGCUUG UGCUAGU 17 17 base pairsnucleic acid single linear 250 CUUGUGCUAG UGUAAUA 17 17 base pairsnucleic acid single linear 251 GCUAGUGUAA UAUAGUG 17 17 base pairsnucleic acid single linear 252 AGUGUAAUAU AGUGUAG 17 17 base pairsnucleic acid single linear 253 UGUAAUAUAG UGUAGUG 17 17 base pairsnucleic acid single linear 254 UAUAGUGUAG UGGUGGC 17 17 base pairsnucleic acid single linear 255 CACAACCUAA UAAGCGC 17 17 base pairsnucleic acid single linear 256 AACCUAAUAA GCGCAUG 17 17 base pairsnucleic acid single linear 257 CAUGAACUAA UUGCUUG 17 17 base pairsnucleic acid single linear 258 GAACUAAUUG CUUGCGU 17 17 base pairsnucleic acid single linear 259 UAAUUGCUUG CGUGUGU 17 17 base pairsnucleic acid single linear 260 GCGUGUGUAG UUAAGUA 17 17 base pairsnucleic acid single linear 261 UGUGUAGUUA AGUACCG 17 17 base pairsnucleic acid single linear 262 GUGUAGUUAA GUACCGA 17 17 base pairsnucleic acid single linear 263 AGUUAAGUAC CGAUCGG 17 17 base pairsnucleic acid single linear 264 GUACCGAUCG GUAAUUU 17 17 base pairsnucleic acid single linear 265 CGAUCGGUAA UUUUAUA 17 17 base pairsnucleic acid single linear 266 UCGGUAAUUU UAUAUUG 17 31 base pairsnucleic acid single linear The letter “N” stands for any base. 267UGGCUGUGGC CUGAUGANGA AAUCGAUCGG U 31 31 base pairs nucleic acid singlelinear The letter “N” stands for any base. 268 GCAGUGAGUU CUGAUGANGAAAUUCCUUCC U 31 31 base pairs nucleic acid single linear The letter “N”stands for any base. 269 GGCUGGCAGU CUGAUGANGA AAGUUUAUUC C 31 31 basepairs nucleic acid single linear The letter “N” stands for any base. 270GACGGAGCAG CUGAUGANGA AACACUUCUC C 31 31 base pairs nucleic acid singlelinear The letter “N” stands for any base. 271 CUGGUGGACG CUGAUGANGAAAGCAGUACA C 31 31 base pairs nucleic acid single linear The letter “N”stands for any base. 272 CGCACUGGUG CUGAUGANGA AACGGAGCAG U 31 31 basepairs nucleic acid single linear The letter “N” stands for any base. 273UCGACGAGAU CUGAUGANGA AAGCAGCCCU G 31 31 base pairs nucleic acid singlelinear The letter “N” stands for any base. 274 UCGUCGACGA CUGAUGANGAAAUGAGCAGC C 31 31 base pairs nucleic acid single linear The letter “N”stands for any base. 275 GGUCGUCGAC CUGAUGANGA AAGAUGAGCA G 31 31 basepairs nucleic acid single linear The letter “N” stands for any base. 276ACUGGUCGUC CUGAUGANGA AACGAGAUGA G 31 31 base pairs nucleic acid singlelinear The letter “N” stands for any base. 277 CAUGCCGAUU CUGAUGANGAAAUCCACUGG U 31 31 base pairs nucleic acid single linear The letter “N”stands for any base. 278 CCAUGCCGAU CUGAUGANGA AAAUCCACUG G 31 31 basepairs nucleic acid single linear The letter “N” stands for any base. 279CCGCCAUGCC CUGAUGANGA AAUUAAUCCA C 31 31 base pairs nucleic acid singlelinear The letter “N” stands for any base. 280 GACGUGGCUA CUGAUGANGAAAGCCGCCAU G 31 31 base pairs nucleic acid single linear The letter “N”stands for any base. 281 GCGACGUGGC CUGAUGANGA AAGAGCCGCC A 31 31 basepairs nucleic acid single linear The letter “N” stands for any base. 282GACGAGCUGC CUGAUGANGA AACGUGGCUA G 31 31 base pairs nucleic acid singlelinear The letter “N” stands for any base. 283 GCGUUGCGAC CUGAUGANGAAAGCUGCGAC G 31 31 base pairs nucleic acid single linear The letter “N”stands for any base. 284 CGCGCGUUGC CUGAUGANGA AACGAGCUGC G 31 31 basepairs nucleic acid single linear The letter “N” stands for any base. 285ACGCGUCCGG CUGAUGANGA AACGCCCAGG C 31 31 base pairs nucleic acid singlelinear The letter “N” stands for any base. 286 GCGGAACGUG CUGAUGANGAAACGCGUCCG G 31 31 base pairs nucleic acid single linear The letter “N”stands for any base. 287 GCCGCGGCGG CUGAUGANGA AACGUGGACG C 31 31 basepairs nucleic acid single linear The letter “N” stands for any base. 288CGCCGCGGCG CUGAUGANGA AAACGUGGAC G 31 31 base pairs nucleic acid singlelinear The letter “N” stands for any base. 289 GUCCGCCGCC CUGAUGANGAAACGCCGUCC G 31 31 base pairs nucleic acid single linear The letter “N”stands for any base. 290 UCCGAAUGCU CUGAUGANGA AAGCGUGUCC G 31 31 basepairs nucleic acid single linear The letter “N” stands for any base. 291CGCUGGUCCG CUGAUGANGA AAUGCUGAGC G 31 31 base pairs nucleic acid singlelinear The letter “N” stands for any base. 292 GCGCUGGUCC CUGAUGANGAAAAUGCUGAG C 31 31 base pairs nucleic acid single linear The letter “N”stands for any base. 293 GCUGGUGCUG CUGAUGANGA AAGCCUGGGC G 31 31 basepairs nucleic acid single linear The letter “N” stands for any base. 294GAGCGACGGG CUGAUGANGA AACCUGGCCC C 31 31 base pairs nucleic acid singlelinear The letter “N” stands for any base. 295 CGAGCGACGG CUGAUGANGAAAACCUGGCC C 31 31 base pairs nucleic acid single linear The letter “N”stands for any base. 296 CACGACGAGC CUGAUGANGA AACGGGAACC U 31 31 basepairs nucleic acid single linear The letter “N” stands for any base. 297CGCACACGAC CUGAUGANGA AAGCGACGGG A 31 31 base pairs nucleic acid singlelinear The letter “N” stands for any base. 298 UGGCGCACAC CUGAUGANGAAACGAGCGAC G 31 31 base pairs nucleic acid single linear The letter “N”stands for any base. 299 CGACGAAGAC CUGAUGANGA AACGUUCAUG C 31 31 basepairs nucleic acid single linear The letter “N” stands for any base. 300CGCCGACGAA CUGAUGANGA AACGACGUUC A 31 31 base pairs nucleic acid singlelinear The letter “N” stands for any base. 301 GGCGCCGACG CUGAUGANGAAAGACGACGU U 31 31 base pairs nucleic acid single linear The letter “N”stands for any base. 302 CGGCGCCGAC CUGAUGANGA AAAGACGACG U 31 31 basepairs nucleic acid single linear The letter “N” stands for any base. 303UCUCGGCGCC CUGAUGANGA AACGAAGACG A 31 31 base pairs nucleic acid singlelinear The letter “N” stands for any base. 304 GGACGUCGCC CUGAUGANGAAAGGCCGCCG G 31 31 base pairs nucleic acid single linear The letter “N”stands for any base. 305 GGCCGCCGAG CUGAUGANGA AACGUCGCCG A 31 31 basepairs nucleic acid single linear The letter “N” stands for any base. 306GCAGGCCGCC CUGAUGANGA AAGGACGUCG C 31 31 base pairs nucleic acid singlelinear The letter “N” stands for any base. 307 AGACGACCAU CUGAUGANGAAACACGGUGC C 31 31 base pairs nucleic acid single linear The letter “N”stands for any base. 308 GGGGAGAGAC CUGAUGANGA AACCAUGACA C 31 31 basepairs nucleic acid single linear The letter “N” stands for any base. 309AGCGGGGAGA CUGAUGANGA AACGACCAUG A 31 31 base pairs nucleic acid singlelinear The letter “N” stands for any base. 310 GUAGCGGGGA CUGAUGANGAAAGACGACCA U 31 31 base pairs nucleic acid single linear The letter “N”stands for any base. 311 UCGUAGCGGG CUGAUGANGA AAGAGACGAC C 31 31 basepairs nucleic acid single linear The letter “N” stands for any base. 312GUACUGGUCG CUGAUGANGA AAGCGGGGAG A 31 31 base pairs nucleic acid singlelinear The letter “N” stands for any base. 313 GGCGUCCUUG CUGAUGANGAAACUGGUCGU A 31 31 base pairs nucleic acid single linear The letter “N”stands for any base. 314 UCUCGGACAC CUGAUGANGA AACGCUGGUG U 31 31 basepairs nucleic acid single linear The letter “N” stands for any base. 315CUUGAUCUCG CUGAUGANGA AACACGACGC U 31 31 base pairs nucleic acid singlelinear The letter “N” stands for any base. 316 CUCCCAUCUU CUGAUGANGAAAUCUCGGAC A 31 31 base pairs nucleic acid single linear The letter “N”stands for any base. 317 GACCGUCUCG CUGAUGANGA AACCUGUCUC C 31 31 basepairs nucleic acid single linear The letter “N” stands for any base. 318GGAAGAACCU CUGAUGANGA AACCGUCUCG U 31 31 base pairs nucleic acid singlelinear The letter “N” stands for any base. 319 GCAGUGGAAG CUGAUGANGAAACCUGACCG U 31 31 base pairs nucleic acid single linear The letter “N”stands for any base. 320 AGCAGUGGAA CUGAUGANGA AAACCUGACC G 31 31 basepairs nucleic acid single linear The letter “N” stands for any base. 321GUAGCAGUGG CUGAUGANGA AAGAACCUGA C 31 31 base pairs nucleic acid singlelinear The letter “N” stands for any base. 322 UGUAGCAGUG CUGAUGANGAAAAGAACCUG A 31 31 base pairs nucleic acid single linear The letter “N”stands for any base. 323 UCCGCGCUUG CUGAUGANGA AAGCAGUGGA A 31 31 basepairs nucleic acid single linear The letter “N” stands for any base. 324GUGGUCAACG CUGAUGANGA AACACGCGGU C 31 31 base pairs nucleic acid singlelinear The letter “N” stands for any base. 325 GGUGGUCAAC CUGAUGANGAAAACACGCGG U 31 31 base pairs nucleic acid single linear The letter “N”stands for any base. 326 GUGGGUGGUC CUGAUGANGA AACGAACACG C 31 31 basepairs nucleic acid single linear The letter “N” stands for any base. 327CCUCUCCAGG CUGAUGANGA AACAGUGGGU G 31 31 base pairs nucleic acid singlelinear The letter “N” stands for any base. 328 CCCUCUCCAG CUGAUGANGAAAACAGUGGG U 31 31 base pairs nucleic acid single linear The letter “N”stands for any base. 329 UCUUUCCCCA CUGAUGANGA AACCCUCUCC A 31 31 basepairs nucleic acid single linear The letter “N” stands for any base. 330GUCUUUCCCC CUGAUGANGA AAACCCUCUC C 31 31 base pairs nucleic acid singlelinear The letter “N” stands for any base. 331 CAGGCCCGUA CUGAUGANGAAAUCUUCUCC U 31 31 base pairs nucleic acid single linear The letter “N”stands for any base. 332 GUCAGGCCCG CUGAUGANGA AAGAUCUUCU C 31 31 basepairs nucleic acid single linear The letter “N” stands for any base. 333GUUGUCCCUG CUGAUGANGA AAGUCCGUUC C 31 31 base pairs nucleic acid singlelinear The letter “N” stands for any base. 334 UAGCAGGCUG CUGAUGANGAAACCGCAGCU G 31 31 base pairs nucleic acid single linear The letter “N”stands for any base. 335 AUAGCAGGCU CUGAUGANGA AAACCGCAGC U 31 31 basepairs nucleic acid single linear The letter “N” stands for any base. 336CUGCCUGGCA CUGAUGANGA AAGCAGGCUG A 31 31 base pairs nucleic acid singlelinear The letter “N” stands for any base. 337 UUGGAGCUUC CUGAUGANGAAAGUGCUGCC U 31 31 base pairs nucleic acid single linear The letter “N”stands for any base. 338 AGGAUCCUUG CUGAUGANGA AAGCUUCAAG U 31 31 basepairs nucleic acid single linear The letter “N” stands for any base. 339UGAGGCUCAG CUGAUGANGA AAUCCUUGGA G 31 31 base pairs nucleic acid singlelinear The letter “N” stands for any base. 340 GGUUGUUGUU CUGAUGANGAAAGGCUCAGG A 31 31 base pairs nucleic acid single linear The letter “N”stands for any base. 341 UCCGGAGAAG CUGAUGANGA AAUGGGUUGU U 31 31 basepairs nucleic acid single linear The letter “N” stands for any base. 342UGGUCCGGAG CUGAUGANGA AAGUAUGGGU U 31 31 base pairs nucleic acid singlelinear The letter “N” stands for any base. 343 AUGGUCCGGA CUGAUGANGAAAAGUAUGGG U 31 31 base pairs nucleic acid single linear The letter “N”stands for any base. 344 GUAUGGUCCG CUGAUGANGA AAGAAGUAUG G 31 31 basepairs nucleic acid single linear The letter “N” stands for any base. 345GUCCUCCCCG CUGAUGANGA AAUGGUCCGG A 31 31 base pairs nucleic acid singlelinear The letter “N” stands for any base. 346 AGACGAACAC CUGAUGANGAAACGUCCUCC C 31 31 base pairs nucleic acid single linear The letter “N”stands for any base. 347 GUUGCAGACG CUGAUGANGA AACACGACGU C 31 31 basepairs nucleic acid single linear The letter “N” stands for any base. 348CGUUGCAGAC CUGAUGANGA AAACACGACG U 31 31 base pairs nucleic acid singlelinear The letter “N” stands for any base. 349 AGUCGUUGCA CUGAUGANGAAACGAACACG A 31 31 base pairs nucleic acid single linear The letter “N”stands for any base. 350 UAGCACGAGA CUGAUGANGA AAGGGCCGGU G 31 31 basepairs nucleic acid single linear The letter “N” stands for any base. 351GGUAGCACGA CUGAUGANGA AAGAGGGCCG G 31 31 base pairs nucleic acid singlelinear The letter “N” stands for any base. 352 GAGGUAGCAC CUGAUGANGAAAGAGAGGGC C 31 31 base pairs nucleic acid single linear The letter “N”stands for any base. 353 GCUCUUGAGG CUGAUGANGA AAGCACGAGA G 31 31 basepairs nucleic acid single linear The letter “N” stands for any base. 354AGUUGCUCUU CUGAUGANGA AAGGUAGCAC G 31 31 base pairs nucleic acid singlelinear The letter “N” stands for any base. 355 GUGGGACUGG CUGAUGANGAAAGUUGCUCU U 31 31 base pairs nucleic acid single linear The letter “N”stands for any base. 356 GAUGCCGUGG CUGAUGANGA AACUGGUAGU U 31 31 basepairs nucleic acid single linear The letter “N” stands for any base. 357CGUCCCUGUA CUGAUGANGA AAUGCCGUGG G 31 31 base pairs nucleic acid singlelinear The letter “N” stands for any base. 358 UGCGUCCCUG CUGAUGANGAAAGAUGCCGU G 31 31 base pairs nucleic acid single linear The letter “N”stands for any base. 359 UGGAUGCAGA CUGAUGANGA AAGCGGUCUU U 31 31 basepairs nucleic acid single linear The letter “N” stands for any base. 360GUGGAUGCAG CUGAUGANGA AAAGCGGUCU U 31 31 base pairs nucleic acid singlelinear The letter “N” stands for any base. 361 UGUGGAUGCA CUGAUGANGAAAAAGCGGUC U 31 31 base pairs nucleic acid single linear The letter “N”stands for any base. 362 AGAUGUUGUG CUGAUGANGA AAUGCAGAAA G 31 31 basepairs nucleic acid single linear The letter “N” stands for any base. 363CCUGGUAGGA CUGAUGANGA AAUGUUGUGG A 31 31 base pairs nucleic acid singlelinear The letter “N” stands for any base. 364 GCCCUGGUAG CUGAUGANGAAAGAUGUUGU G 31 31 base pairs nucleic acid single linear The letter “N”stands for any base. 365 CCGGCCCUGG CUGAUGANGA AAGGAGAUGU U 31 31 basepairs nucleic acid single linear The letter “N” stands for any base. 366GGAGAAGGCG CUGAUGANGA AACCGGCCCU G 31 31 base pairs nucleic acid singlelinear The letter “N” stands for any base. 367 CGGAGAAGGC CUGAUGANGAAAACCGGCCC U 31 31 base pairs nucleic acid single linear The letter “N”stands for any base. 368 GUAGUCGGAG CUGAUGANGA AAGGCGAACC G 31 31 basepairs nucleic acid single linear The letter “N” stands for any base. 369GGUAGUCGGA CUGAUGANGA AAAGGCGAAC C 31 31 base pairs nucleic acid singlelinear The letter “N” stands for any base. 370 CGGGUAGUCG CUGAUGANGAAAGAAGGCGA A 31 31 base pairs nucleic acid single linear The letter “N”stands for any base. 371 CAGCUCCGGG CUGAUGANGA AAGUCGGAGA A 31 31 basepairs nucleic acid single linear The letter “N” stands for any base. 372AUCUCUCCGG CUGAUGANGA AAGGUUCAGC U 31 31 base pairs nucleic acid singlelinear The letter “N” stands for any base. 373 GGACGACUUG CUGAUGANGAAAUCUCUCCG G 31 31 base pairs nucleic acid single linear The letter “N”stands for any base. 374 AGGACGACUU CUGAUGANGA AAAUCUCUCC G 31 31 basepairs nucleic acid single linear The letter “N” stands for any base. 375AUCGAAGGAC CUGAUGANGA AACUUGAAUC U 31 31 base pairs nucleic acid singlelinear The letter “N” stands for any base. 376 GAAAUCGAAG CUGAUGANGAAACGACUUGA A 31 31 base pairs nucleic acid single linear The letter “N”stands for any base. 377 GAUGAAAUCG CUGAUGANGA AAGGACGACU U 31 31 basepairs nucleic acid single linear The letter “N” stands for any base. 378CGAUGAAAUC CUGAUGANGA AAAGGACGAC U 31 31 base pairs nucleic acid singlelinear The letter “N” stands for any base. 379 CCGUCGAUGA CUGAUGANGAAAUCGAAGGA C 31 31 base pairs nucleic acid single linear The letter “N”stands for any base. 380 GCCGUCGAUG CUGAUGANGA AAAUCGAAGG A 31 31 basepairs nucleic acid single linear The letter “N” stands for any base. 381AGCCGUCGAU CUGAUGANGA AAAAUCGAAG G 31 31 base pairs nucleic acid singlelinear The letter “N” stands for any base. 382 CGUAGCCGUC CUGAUGANGAAAUGAAAUCG A 31 31 base pairs nucleic acid single linear The letter “N”stands for any base. 383 GGGCUUCUCG CUGAUGANGA AAGCCGUCGA U 31 31 basepairs nucleic acid single linear The letter “N” stands for any base. 384UCAUCCAGUU CUGAUGANGA AAUCUUCCGG C 31 31 base pairs nucleic acid singlelinear The letter “N” stands for any base. 385 CGGCCUCGAG CUGAUGANGAAAUCCCGGCC U 31 31 base pairs nucleic acid single linear The letter “N”stands for any base. 386 UGUCGGCCUC CUGAUGANGA AAGGAUCCCG G 31 31 basepairs nucleic acid single linear The letter “N” stands for any base. 387UGACGGUGAG CUGAUGANGA AACCCUGUCG G 31 31 base pairs nucleic acid singlelinear The letter “N” stands for any base. 388 GGCUGACGGU CUGAUGANGAAAGGACCCUG U 31 31 base pairs nucleic acid single linear The letter “N”stands for any base. 389 AGUAGGGGCU CUGAUGANGA AACGGUGAGG A 31 31 basepairs nucleic acid single linear The letter “N” stands for any base. 390CUCGGCGUAG CUGAUGANGA AAGGGGCUGA C 31 31 base pairs nucleic acid singlelinear The letter “N” stands for any base. 391 CUCCUCGGCG CUGAUGANGAAAGUAGGGGC U 31 31 base pairs nucleic acid single linear The letter “N”stands for any base. 392 UGCCGGAGAU CUGAUGANGA AAGCUCCUCG G 31 31 basepairs nucleic acid single linear The letter “N” stands for any base. 393CGAUGCCGGA CUGAUGANGA AAUGAGCUCC U 31 31 base pairs nucleic acid singlelinear The letter “N” stands for any base. 394 GGCGAUGCCG CUGAUGANGAAAGAUGAGCU C 31 31 base pairs nucleic acid single linear The letter “N”stands for any base. 395 AGCCCCUGGC CUGAUGANGA AAUGCCGGAG A 31 31 basepairs nucleic acid single linear The letter “N” stands for any base. 396UGAUGUUGUC CUGAUGANGA AAGCUCGCAG C 31 31 base pairs nucleic acid singlelinear The letter “N” stands for any base. 397 UGAGGCGCAU CUGAUGANGAAAUGUUGUCG A 31 31 base pairs nucleic acid single linear The letter “N”stands for any base. 398 UGAUGCCGGU CUGAUGANGA AAGGCGCAUG A 31 31 basepairs nucleic acid single linear The letter “N” stands for any base. 399CGAUGCCGGU CUGAUGANGA AAUGCCGGUG A 31 31 base pairs nucleic acid singlelinear The letter “N” stands for any base. 400 UGCCGUUGAC CUGAUGANGAAAUGCCGGUG A 31 31 base pairs nucleic acid single linear The letter “N”stands for any base. 401 CCAUGCCGUU CUGAUGANGA AACGAUGCCG G 31 31 basepairs nucleic acid single linear The letter “N” stands for any base. 402CCCACUCGCU CUGAUGANGA AACGUCCAUG C 31 31 base pairs nucleic acid singlelinear The letter “N” stands for any base. 403 CACGGCGAUG CUGAUGANGAAACUUGUCCC U 31 31 base pairs nucleic acid single linear The letter “N”stands for any base. 404 ACUUCACGGC CUGAUGANGA AAUGUACUUG U 31 31 basepairs nucleic acid single linear The letter “N” stands for any base. 405CGACACGUCG CUGAUGANGA AACUUCACGG C 31 31 base pairs nucleic acid singlelinear The letter “N” stands for any base. 406 CACGGCCGUC CUGAUGANGAAACACGUCGU A 31 31 base pairs nucleic acid single linear The letter “N”stands for any base. 407 CCGGGAGCCC CUGAUGANGA AACCUCCGCC U 31 31 basepairs nucleic acid single linear The letter “N” stands for any base. 408GGUCCACCGG CUGAUGANGA AAGCCCGACC U 31 31 base pairs nucleic acid singlelinear The letter “N” stands for any base. 409 CCACCAGCGG CUGAUGANGAAAUGUUCCGG U 31 31 base pairs nucleic acid single linear The letter “N”stands for any base. 410 CCUGCCGAUG CUGAUGANGA AACGCCACCA G 31 31 basepairs nucleic acid single linear The letter “N” stands for any base. 411GCCUGCCGAU CUGAUGANGA AAACGCCACC A 31 31 base pairs nucleic acid singlelinear The letter “N” stands for any base. 412 CCAGCCUGCC CUGAUGANGAAAUGAACGCC A 31 31 base pairs nucleic acid single linear The letter “N”stands for any base. 413 CGGCCGCCAU CUGAUGANGA AACGUCGGGU C 31 31 basepairs nucleic acid single linear The letter “N” stands for any base. 414UGAGCUGCGG CUGAUGANGA AAUGGCGGCC G 31 31 base pairs nucleic acid singlelinear The letter “N” stands for any base. 415 CCAUCUCCAU CUGAUGANGAAAGCUGCGGG A 31 31 base pairs nucleic acid single linear The letter “N”stands for any base. 416 CCAGCAGAAC CUGAUGANGA AAUCUGCACG U 31 31 basepairs nucleic acid single linear The letter “N” stands for any base. 417UGCCCAGCAG CUGAUGANGA AACGAUCUGC A 31 31 base pairs nucleic acid singlelinear The letter “N” stands for any base. 418 GUGCCCAGCA CUGAUGANGAAAACGAUCUG C 31 31 base pairs nucleic acid single linear The letter “N”stands for any base. 419 CAUGCGCUCG CUGAUGANGA AACUUCUUCU U 31 31 basepairs nucleic acid single linear The letter “N” stands for any base. 420GCAUGCGCUC CUGAUGANGA AAACUUCUUC U 31 31 base pairs nucleic acid singlelinear The letter “N” stands for any base. 421 CGGCGCUCAU CUGAUGANGAAAGCAUGCGC U 31 31 base pairs nucleic acid single linear The letter “N”stands for any base. 422 CUUGCCUGGG CUGAUGANGA AACUUCUCCU C 31 31 basepairs nucleic acid single linear The letter “N” stands for any base. 423CCUUGCCUGG CUGAUGANGA AAACUUCUCC U 31 31 base pairs nucleic acid singlelinear The letter “N” stands for any base. 424 CGUUGAACUU CUGAUGANGAAACCACGGCG C 31 31 base pairs nucleic acid single linear The letter “N”stands for any base. 425 CGCCGCGUUG CUGAUGANGA AACUUGACCA C 31 31 basepairs nucleic acid single linear The letter “N” stands for any base. 426GCGCCGCGUU CUGAUGANGA AAACUUGACC A 31 31 base pairs nucleic acid singlelinear The letter “N” stands for any base. 427 CGCCGGCCAU CUGAUGANGAAAUGUGGUGC G 31 31 base pairs nucleic acid single linear The letter “N”stands for any base. 428 UGGUGACGGC CUGAUGANGA AAGCACGUCG G 31 31 basepairs nucleic acid single linear The letter “N” stands for any base. 429AGCGGCUGGU CUGAUGANGA AACGGCGAGC A 31 31 base pairs nucleic acid singlelinear The letter “N” stands for any base. 430 GCAGGGCUCG CUGAUGANGAAAGCGGCUGG U 31 31 base pairs nucleic acid single linear The letter “N”stands for any base. 431 CGCAGGGCUC CUGAUGANGA AAAGCGGCUG G 31 31 basepairs nucleic acid single linear The letter “N” stands for any base. 432GCAGCUGGAU CUGAUGANGA AAGGCCGCAG G 31 31 base pairs nucleic acid singlelinear The letter “N” stands for any base. 433 CCUGCAGCUG CUGAUGANGAAAUGAGGCCG C 31 31 base pairs nucleic acid single linear The letter “N”stands for any base. 434 GGGCGUUCCG CUGAUGANGA AAUCGCAUCC C 31 31 basepairs nucleic acid single linear The letter “N” stands for any base. 435UCCACCGGUG CUGAUGANGA AACGCGCAGG C 31 31 base pairs nucleic acid singlelinear The letter “N” stands for any base. 436 UGGUGUCGAC CUGAUGANGAAAGUCCACCG G 31 31 base pairs nucleic acid single linear The letter “N”stands for any base. 437 UGAUGGUGUC CUGAUGANGA AACGAGUCCA C 31 31 basepairs nucleic acid single linear The letter “N” stands for any base. 438UGCCUUCGAU CUGAUGANGA AAUGGUGUCG A 31 31 base pairs nucleic acid singlelinear The letter “N” stands for any base. 439 UCUUGCCUUC CUGAUGANGAAAUGAUGGUG U 31 31 base pairs nucleic acid single linear The letter “N”stands for any base. 440 GCCCAUGUGG CUGAUGANGA AACCCGGUCU U 31 31 basepairs nucleic acid single linear The letter “N” stands for any base. 441GGCCCAUGUG CUGAUGANGA AAACCCGGUC U 31 31 base pairs nucleic acid singlelinear The letter “N” stands for any base. 442 AGUCGACGCU CUGAUGANGAAAGGCGGCCC A 31 31 base pairs nucleic acid single linear The letter “N”stands for any base. 443 CGUUGCAGUC CUGAUGANGA AACGCUGAGG C 31 31 basepairs nucleic acid single linear The letter “N” stands for any base. 444CCGGCUCCAC CUGAUGANGA AACGUUGCAG U 31 31 base pairs nucleic acid singlelinear The letter “N” stands for any base. 445 CCACCUUCUU CUGAUGANGAAACGUCCGCC G 31 31 base pairs nucleic acid single linear The letter “N”stands for any base. 446 GGCGCGCUGC CUGAUGANGA AAGGUGGUGG C 31 31 basepairs nucleic acid single linear The letter “N” stands for any base. 447CGACCACCUU CUGAUGANGA AAUGGCGCGC U 31 31 base pairs nucleic acid singlelinear The letter “N” stands for any base. 448 CCGGCGUGCC CUGAUGANGAAACCACCUUG A 31 31 base pairs nucleic acid single linear The letter “N”stands for any base. 449 CAUCUCCUCG CUGAUGANGA AACGCCGGCG U 31 31 basepairs nucleic acid single linear The letter “N” stands for any base. 450AGAGAUCCUG CUGAUGANGA AAUCAUGCAG U 31 31 base pairs nucleic acid singlelinear The letter “N” stands for any base. 451 UUCCAGGAGA CUGAUGANGAAAUCCUGGAU C 31 31 base pairs nucleic acid single linear The letter “N”stands for any base. 452 CCUUCCAGGA CUGAUGANGA AAGAUCCUGG A 31 31 basepairs nucleic acid single linear The letter “N” stands for any base. 453GCCCUUCCAG CUGAUGANGA AAGAGAUCCU G 31 31 base pairs nucleic acid singlelinear The letter “N” stands for any base. 454 CCCCGAGGCU CUGAUGANGAAAGCAGCACG U 31 31 base pairs nucleic acid single linear The letter “N”stands for any base. 455 CGGCGACCCC CUGAUGANGA AAGGCUGAGC A 31 31 basepairs nucleic acid single linear The letter “N” stands for any base. 456CGCCGCCGGC CUGAUGANGA AACCCCGAGG C 31 31 base pairs nucleic acid singlelinear The letter “N” stands for any base. 457 CCUCGCCUUC CUGAUGANGAAACCCCUGGC U 31 31 base pairs nucleic acid single linear The letter “N”stands for any base. 458 CGAGCGGCGC CUGAUGANGA AAUCUCCUCG C 31 31 basepairs nucleic acid single linear The letter “N” stands for any base. 459UCUCCUUGGC CUGAUGANGA AAGCGGCGCG A 31 31 base pairs nucleic acid singlelinear The letter “N” stands for any base. 460 CUGCAGGCCG CUGAUGANGAAACUCUUCAG G 31 31 base pairs nucleic acid single linear The letter “N”stands for any base. 461 CCUGCAGGCC CUGAUGANGA AAACUCUUCA G 31 31 basepairs nucleic acid single linear The letter “N” stands for any base. 462CCACGCGCGA CUGAUGANGA AAUCAGGGGG C 31 31 base pairs nucleic acid singlelinear The letter “N” stands for any base. 463 CACCACGCGC CUGAUGANGAAAGAUCAGGG G 31 31 base pairs nucleic acid single linear The letter “N”stands for any base. 464 AAGAUGUCCC CUGAUGANGA AACAUGUUUG C 31 31 basepairs nucleic acid single linear The letter “N” stands for any base. 465UAUAUAAGAA CUGAUGANGA AAUGUCCCAA C 31 31 base pairs nucleic acid singlelinear The letter “N” stands for any base. 466 CAUAUAUAAG CUGAUGANGAAAGAUGUCCC A 31 31 base pairs nucleic acid single linear The letter “N”stands for any base. 467 GCAUAUAUAA CUGAUGANGA AAAGAUGUCC C 31 31 basepairs nucleic acid single linear The letter “N” stands for any base. 468CAGCAUAUAU CUGAUGANGA AAGAAGAUGU C 31 31 base pairs nucleic acid singlelinear The letter “N” stands for any base. 469 ACAGCAUAUA CUGAUGANGAAAAGAAGAUG U 31 31 base pairs nucleic acid single linear The letter “N”stands for any base. 470 AAACAGCAUA CUGAUGANGA AAUAAGAAGA U 31 31 basepairs nucleic acid single linear The letter “N” stands for any base. 471CGAAACAGCA CUGAUGANGA AAUAUAAGAA G 31 31 base pairs nucleic acid singlelinear The letter “N” stands for any base. 472 ACAUAAACGA CUGAUGANGAAACAGCAUAU A 31 31 base pairs nucleic acid single linear The letter “N”stands for any base. 473 CACAUAAACG CUGAUGANGA AAACAGCAUA U 31 31 basepairs nucleic acid single linear The letter “N” stands for any base. 474UCACAUAAAC CUGAUGANGA AAAACAGCAU A 31 31 base pairs nucleic acid singlelinear The letter “N” stands for any base. 475 AUAUCACAUA CUGAUGANGAAACGAAACAG C 31 31 base pairs nucleic acid single linear The letter “N”stands for any base. 476 CAUAUCACAU CUGAUGANGA AAACGAAACA G 31 31 basepairs nucleic acid single linear The letter “N” stands for any base. 477CCAUAUCACA CUGAUGANGA AAAACGAAAC A 31 31 base pairs nucleic acid singlelinear The letter “N” stands for any base. 478 UACUUGUCCA CUGAUGANGAAAUCACAUAA A 31 31 base pairs nucleic acid single linear The letter “N”stands for any base. 479 CAGCUACACA CUGAUGANGA AACUUGUCCA U 31 31 basepairs nucleic acid single linear The letter “N” stands for any base. 480AGCAAGCAGC CUGAUGANGA AACACAUACU U 31 31 base pairs nucleic acid singlelinear The letter “N” stands for any base. 481 UAGCACAAGC CUGAUGANGAAAGCAGCUAC A 31 31 base pairs nucleic acid single linear The letter “N”stands for any base. 482 ACACUAGCAC CUGAUGANGA AAGCAAGCAG C 31 31 basepairs nucleic acid single linear The letter “N” stands for any base. 483UAUAUUACAC CUGAUGANGA AAGCACAAGC A 31 31 base pairs nucleic acid singlelinear The letter “N” stands for any base. 484 UACACUAUAU CUGAUGANGAAACACUAGCA C 31 31 base pairs nucleic acid single linear The letter “N”stands for any base. 485 CACUACACUA CUGAUGANGA AAUUACACUA G 31 31 basepairs nucleic acid single linear The letter “N” stands for any base. 486ACCACUACAC CUGAUGANGA AAUAUUACAC U 31 31 base pairs nucleic acid singlelinear The letter “N” stands for any base. 487 UGGCCACCAC CUGAUGANGAAACACUAUAU U 31 31 base pairs nucleic acid single linear The letter “N”stands for any base. 488 AUGCGCUUAU CUGAUGANGA AAGGUUGUGC C 31 31 basepairs nucleic acid single linear The letter “N” stands for any base. 489UUCAUGCGCU CUGAUGANGA AAUUAGGUUG U 31 31 base pairs nucleic acid singlelinear The letter “N” stands for any base. 490 CGCAAGCAAU CUGAUGANGAAAGUUCAUGC G 31 31 base pairs nucleic acid single linear The letter “N”stands for any base. 491 ACACGCAAGC CUGAUGANGA AAUUAGUUCA U 31 31 basepairs nucleic acid single linear The letter “N” stands for any base. 492CUACACACGC CUGAUGANGA AAGCAAUUAG U 31 31 base pairs nucleic acid singlelinear The letter “N” stands for any base. 493 GGUACUUAAC CUGAUGANGAAACACACGCA A 31 31 base pairs nucleic acid single linear The letter “N”stands for any base. 494 AUCGGUACUU CUGAUGANGA AACUACACAC G 31 31 basepairs nucleic acid single linear The letter “N” stands for any base. 495GAUCGGUACU CUGAUGANGA AAACUACACA C 31 31 base pairs nucleic acid singlelinear The letter “N” stands for any base. 496 UACCGAUCGG CUGAUGANGAAACUUAACUA C 31 31 base pairs nucleic acid single linear The letter “N”stands for any base. 497 UAAAAUUACC CUGAUGANGA AAUCGGUACU U 31 31 basepairs nucleic acid single linear The letter “N” stands for any base. 498AAUAUAAAAU CUGAUGANGA AACCGAUCGG U 31 31 base pairs nucleic acid singlelinear The letter “N” stands for any base. 499 CGCAAUAUAA CUGAUGANGAAAUUACCGAU C 31 31 base pairs nucleic acid single linear The letter “N”stands for any base. 500 UCGCAAUAUA CUGAUGANGA AAAUUACCGA U 31 31 basepairs nucleic acid single linear The letter “N” stands for any base. 501CUCGCAAUAU CUGAUGANGA AAAAUUACCG A 31 31 base pairs nucleic acid singlelinear The letter “N” stands for any base. 502 ACUCGCAAUA CUGAUGANGAAAAAAUUACC G 31 31 base pairs nucleic acid single linear The letter “N”stands for any base. 503 UUACUCGCAA CUGAUGANGA AAUAAAAUUA C 31 31 basepairs nucleic acid single linear The letter “N” stands for any base. 504AUUUACUCGC CUGAUGANGA AAUAUAAAAU U 31 31 base pairs nucleic acid singlelinear The letter “N” stands for any base. 505 UCCAUUUAUU CUGAUGANGAAACUCGCAAU A 31 31 base pairs nucleic acid single linear The letter “N”stands for any base. 506 CAGGUCCAUU CUGAUGANGA AAUUUACUCG C 31 31 basepairs nucleic acid single linear The letter “N” stands for any base. 507UUUCCACCAC CUGAUGANGA AACAGGUCCA U 31 52 base pairs nucleic acid singlelinear 508 CUCCUGGCAG AAGUCGACCA GAGAAACACA CGUUGUGGUA CAUUACCUGG UA 5216 base pairs nucleic acid single linear 509 CGACAGCCGC CAGGAG 16 52base pairs nucleic acid single linear 510 CCCUGCCGAG AAGUGCACCAGAGAAACACA CGUUGUGGUA CAUUACCUGG UA 52 16 base pairs nucleic acid singlelinear 511 GCACCGCCCG GCAGGG 16 52 base pairs nucleic acid single linear512 GUCGCCGAAG AAGCCGACCA GAGAAACACA CGUUGUGGUA CAUUACCUGG UA 52 16 basepairs nucleic acid single linear 513 CGGCGGCCUC GGCGAC 16 52 base pairsnucleic acid single linear 514 CGGCGGCAAG AAGCCGACCA GAGAAACACACGUUGUGGUA CAUUACCUGG UA 52 16 base pairs nucleic acid single linear 515CGGCGGCCUG CCGCCG 16 52 base pairs nucleic acid single linear 516CCAUGGCCAG AAGCAGACCA GAGAAACACA CGUUGUGGUA CAUUACCUGG UA 52 16 basepairs nucleic acid single linear 517 CUGCCGCCGG CCAUGG 16 52 base pairsnucleic acid single linear 518 UCUCCAGGAG AAGUGGACCA GAGAAACACACGUUGUGGUA CAUUACCUGG UA 52 16 base pairs nucleic acid single linear 519CCACUGUUCC UGGAGA 16 52 base pairs nucleic acid single linear 520UCCCUGUAAG AAGUUCACCA GAGAAACACA CGUUGUGGUA CAUUACCUGG UA 52 16 basepairs nucleic acid single linear 521 GAACGGACUA CAGGGA 16 52 base pairsnucleic acid single linear 522 GCAGGCUGAG AAGCAGACCA GAGAAACACACGUUGUGGUA CAUUACCUGG UA 52 16 base pairs nucleic acid single linear 523CUGCGGUUCA GCCUGC 16 52 base pairs nucleic acid single linear 524GCCUCCACAG AAGUCGACCA GAGAAACACA CGUUGUGGUA CAUUACCUGG UA 52 16 basepairs nucleic acid single linear 525 CGACGGCCGU GGAGGC 16 52 base pairsnucleic acid single linear 526 GGGAUGGCAG AAGCCAACCA GAGAAACACACGUUGUGGUA CAUUACCUGG UA 52 16 base pairs nucleic acid single linear 527UGGCGGCCGC CAUCCC 16 52 base pairs nucleic acid single linear 528GCGAGCACAG AAGCGCACCA GAGAAACACA CGUUGUGGUA CAUUACCUGG UA 52 16 basepairs nucleic acid single linear 529 GCGCCGACGU GCUCGC 16 52 base pairsnucleic acid single linear 530 CUGGAUGAAG AAGCAGACCA GAGAAACACACGUUGUGGUA CAUUACCUGG UA 52 16 base pairs nucleic acid single linear 531CUGCGGCCUC AUCCAG 16 52 base pairs nucleic acid single linear 532GACGCUGAAG AAGCCCACCA GAGAAACACA CGUUGUGGUA CAUUACCUGG UA 52 16 basepairs nucleic acid single linear 533 GGGCCGCCUC AGCGUC 16 52 base pairsnucleic acid single linear 534 UUCUUGACAG AAGCCGACCA GAGAAACACACGUUGUGGUA CAUUACCUGG UA 52 16 base pairs nucleic acid single linear 535CGGCGGACGU CAAGAA 16 52 base pairs nucleic acid single linear 536AUAAACGAAG AAGCAUACCA GAGAAACACA CGUUGUGGUA CAUUACCUGG UA 52 16 basepairs nucleic acid single linear 537 AUGCUGUUUC GUUUAU 16 54 base pairsnucleic acid single linear 538 GUCGCCUCAG AAGGUGGUAC CAGAGAAACACACGUUGUGG UACAUUACCU GGUA 54 18 base pairs nucleic acid single linear539 ACCACCCGCC GAGGCGAC 18 54 base pairs nucleic acid single linear 540CUCCUGGCAG AAGUCGCGAC CAGAGAAACA CACGUUGUGG UACAUUACCU GGUA 54 18 basepairs nucleic acid single linear 541 CGCGACAGCC GCCAGGAG 18 54 basepairs nucleic acid single linear 542 GUGGACGGAG AAGUACACAC CAGAGAAACACACGUUGUGG UACAUUACCU GGUA 54 18 base pairs nucleic acid single linear543 GUGUACUGCU CCGUCCAC 18 54 base pairs nucleic acid single linear 544CACUGGUGAG AAGAGCAGAC CAGAGAAACA CACGUUGUGG UACAUUACCU GGUA 54 18 basepairs nucleic acid single linear 545 CUGCUCCGUC CACCAGUG 18 54 basepairs nucleic acid single linear 546 CCCUGCCGAG AAGUGCGCAC CAGAGAAACACACGUUGUGG UACAUUACCU GGUA 54 18 base pairs nucleic acid single linear547 GCGCACCGCC CGGCAGGG 18 54 base pairs nucleic acid single linear 548ACGAGAUGAG AAGCCCUGAC CAGAGAAACA CACGUUGUGG UACAUUACCU GGUA 54 18 basepairs nucleic acid single linear 549 CAGGGCUGCU CAUCUCGU 18 54 basepairs nucleic acid single linear 550 GUGGCUAGAG AAGCCAUGAC CAGAGAAACACACGUUGUGG UACAUUACCU GGUA 54 18 base pairs nucleic acid single linear551 CAUGGCGGCU CUAGCCAC 18 54 base pairs nucleic acid single linear 552UUGCGACGAG AAGCGACGAC CAGAGAAACA CACGUUGUGG UACAUUACCU GGUA 54 18 basepairs nucleic acid single linear 553 CGUCGCAGCU CGUCGCAA 18 54 basepairs nucleic acid single linear 554 GACGCCCAAG AAGGCGCGAC CAGAGAAACACACGUUGUGG UACAUUACCU GGUA 54 18 base pairs nucleic acid single linear555 CGCGCCGGCC UGGGCGUC 18 54 base pairs nucleic acid single linear 556GUGGACGCAG AAGGGACGAC CAGAGAAACA CACGUUGUGG UACAUUACCU GGUA 54 18 basepairs nucleic acid single linear 557 CGUCCCGGAC GCGUCCAC 18 54 basepairs nucleic acid single linear 558 GGCGCCGCAG AAGAACGUAC CAGAGAAACACACGUUGUGG UACAUUACCU GGUA 54 18 base pairs nucleic acid single linear559 ACGUUCCGCC GCGGCGCC 18 54 base pairs nucleic acid single linear 560CCGACGCCAG AAGGCCCCAC CAGAGAAACA CACGUUGUGG UACAUUACCU GGUA 54 18 basepairs nucleic acid single linear 561 GGGGCCGGAC GGCGUCGG 18 54 basepairs nucleic acid single linear 562 GCGCGCUGAG AAGAAUGCAC CAGAGAAACACACGUUGUGG UACAUUACCU GGUA 54 18 base pairs nucleic acid single linear563 GCAUUCGGAC CAGCGCGC 18 54 base pairs nucleic acid single linear 564CGACGAGCAG AAGGAACCAC CAGAGAAACA CACGUUGUGG UACAUUACCU GGUA 54 18 basepairs nucleic acid single linear 565 GGUUCCCGUC GCUCGUCG 18 54 basepairs nucleic acid single linear 566 GUCGCCGAAG AAGCCGGUAC CAGAGAAACACACGUUGUGG UACAUUACCU GGUA 54 18 base pairs nucleic acid single linear567 ACCGGCGGCC UCGGCGAC 18 54 base pairs nucleic acid single linear 568CGGCGGCAAG AAGCCGAGAC CAGAGAAACA CACGUUGUGG UACAUUACCU GGUA 54 18 basepairs nucleic acid single linear 569 CUCGGCGGCC UGCCGCCG 18 54 basepairs nucleic acid single linear 570 UGGCCGGCAG AAGGCCGCAC CAGAGAAACACACGUUGUGG UACAUUACCU GGUA 54 18 base pairs nucleic acid single linear571 GCGGCCUGCC GCCGGCCA 18 54 base pairs nucleic acid single linear 572CCAUGGCCAG AAGCAGGCAC CAGAGAAACA CACGUUGUGG UACAUUACCU GGUA 54 18 basepairs nucleic acid single linear 573 GCCUGCCGCC GGCCAUGG 18 54 basepairs nucleic acid single linear 574 UCUCCAGGAG AAGUGGGUAC CAGAGAAACACACGUUGUGG UACAUUACCU GGUA 54 18 base pairs nucleic acid single linear575 ACCCACUGUU CCUGGAGA 18 54 base pairs nucleic acid single linear 576GUUCCAGCAG AAGGCCCGAC CAGAGAAACA CACGUUGUGG UACAUUACCU GGUA 54 18 basepairs nucleic acid single linear 577 CGGGCCUGAC GCUGGAAC 18 54 basepairs nucleic acid single linear 578 UCCCUGUAAG AAGUUCCAAC CAGAGAAACACACGUUGUGG UACAUUACCU GGUA 54 18 base pairs nucleic acid single linear579 UGGAACGGAC UACAGGGA 18 54 base pairs nucleic acid single linear 580UGAACCGCAG AAGGUUGUAC CAGAGAAACA CACGUUGUGG UACAUUACCU GGUA 54 18 basepairs nucleic acid single linear 581 ACAACCAGCU GCGGUUCA 18 54 basepairs nucleic acid single linear 582 GCAGGCUGAG AAGCAGCUAC CAGAGAAACACACGUUGUGG UACAUUACCU GGUA 54 18 base pairs nucleic acid single linear583 AGCUGCGGUU CAGCCUGC 18 54 base pairs nucleic acid single linear 584GCAUAGCAAG AAGAACCGAC CAGAGAAACA CACGUUGUGG UACAUUACCU GGUA 54 18 basepairs nucleic acid single linear 585 CGGUUCAGCC UGCUAUGC 18 54 basepairs nucleic acid single linear 586 CCCGUAUGAG AAGGAGAAAC CAGAGAAACACACGUUGUGG UACAUUACCU GGUA 54 18 base pairs nucleic acid single linear587 UUCUCCGGAC CAUACGGG 18 54 base pairs nucleic acid single linear 588CGAGAGAGAG AAGGUGUGAC CAGAGAAACA CACGUUGUGG UACAUUACCU GGUA 54 18 basepairs nucleic acid single linear 589 CACACCGGCC CUCUCUCG 18 54 basepairs nucleic acid single linear 590 UGCCGUGGAG AAGGUAGUAC CAGAGAAACACACGUUGUGG UACAUUACCU GGUA 54 18 base pairs nucleic acid single linear591 ACUACCAGUC CCACGGCA 18 54 base pairs nucleic acid single linear 592AUGCAGAAAG AAGUCUUUAC CAGAGAAACA CACGUUGUGG UACAUUACCU GGUA 54 18 basepairs nucleic acid single linear 593 AAAGACCGCU UUCUGCAU 18 54 basepairs nucleic acid single linear 594 AGAAGGCGAG AAGGCCCUAC CAGAGAAACACACGUUGUGG UACAUUACCU GGUA 54 18 base pairs nucleic acid single linear595 AGGGCCGGUU CGCCUUCU 18 54 base pairs nucleic acid single linear 596UCCGGGUAAG AAGAGAAGAC CAGAGAAACA CACGUUGUGG UACAUUACCU GGUA 54 18 basepairs nucleic acid single linear 597 CUUCUCCGAC UACCCGGA 18 54 basepairs nucleic acid single linear 598 GUAGUAGGAG AAGACGGUAC CAGAGAAACACACGUUGUGG UACAUUACCU GGUA 54 18 base pairs nucleic acid single linear599 ACCGUCAGCC CCUACUAC 18 54 base pairs nucleic acid single linear 600GCCUCCACAG AAGUCGACAC CAGAGAAACA CACGUUGUGG UACAUUACCU GGUA 54 18 basepairs nucleic acid single linear 601 GUCGACGGCC GUGGAGGC 18 54 basepairs nucleic acid single linear 602 ACGCCACCAG AAGGAUGUAC CAGAGAAACACACGUUGUGG UACAUUACCU GGUA 54 18 base pairs nucleic acid single linear603 ACAUCCCGCU GGUGGCGU 18 54 base pairs nucleic acid single linear 604GCCAUGACAG AAGGUCCCAC CAGAGAAACA CACGUUGUGG UACAUUACCU GGUA 54 18 basepairs nucleic acid single linear 605 GGGACCCGAC GUCAUGGC 18 54 basepairs nucleic acid single linear 606 GGGAUGGCAG AAGCCAUGAC CAGAGAAACACACGUUGUGG UACAUUACCU GGUA 54 18 base pairs nucleic acid single linear607 CAUGGCGGCC GCCAUCCC 18 54 base pairs nucleic acid single linear 608UCUCCAUGAG AAGCGGGAAC CAGAGAAACA CACGUUGUGG UACAUUACCU GGUA 54 18 basepairs nucleic acid single linear 609 UCCCGCAGCU CAUGGAGA 18 54 basepairs nucleic acid single linear 610 GCAGAACGAG AAGCACGUAC CAGAGAAACACACGUUGUGG UACAUUACCU GGUA 54 18 base pairs nucleic acid single linear611 ACGUGCAGAU CGUUCUGC 18 54 base pairs nucleic acid single linear 612CCGUGCCCAG AAGAACGAAC CAGAGAAACA CACGUUGUGG UACAUUACCU GGUA 54 18 basepairs nucleic acid single linear 613 UCGUUCUGCU GGGCACGG 18 54 basepairs nucleic acid single linear 614 GCGAGCACAG AAGCGCCGAC CAGAGAAACACACGUUGUGG UACAUUACCU GGUA 54 18 base pairs nucleic acid single linear615 CGGCGCCGAC GUGCUCGC 18 54 base pairs nucleic acid single linear 616CUCGAAGCAG AAGGUGACAC CAGAGAAACA CACGUUGUGG UACAUUACCU GGUA 54 18 basepairs nucleic acid single linear 617 GUCACCAGCC GCUUCGAG 18 54 basepairs nucleic acid single linear 618 GGGCUCGAAG AAGCUGGUAC CAGAGAAACACACGUUGUGG UACAUUACCU GGUA 54 18 base pairs nucleic acid single linear619 ACCAGCCGCU UCGAGCCC 18 54 base pairs nucleic acid single linear 620CUGGAUGAAG AAGCAGGGAC CAGAGAAACA CACGUUGUGG UACAUUACCU GGUA 54 18 basepairs nucleic acid single linear 621 CCCUGCGGCC UCAUCCAG 18 54 basepairs nucleic acid single linear 622 UCCCCUGCAG AAGGAUGAAC CAGAGAAACACACGUUGUGG UACAUUACCU GGUA 54 18 base pairs nucleic acid single linear623 UCAUCCAGCU GCAGGGGA 18 54 base pairs nucleic acid single linear 624GACGCUGAAG AAGCCCAUAC CAGAGAAACA CACGUUGUGG UACAUUACCU GGUA 54 18 basepairs nucleic acid single linear 625 AUGGGCCGCC UCAGCGUC 18 54 basepairs nucleic acid single linear 626 UUCUUGACAG AAGCCGGCAC CAGAGAAACACACGUUGUGG UACAUUACCU GGUA 54 18 base pairs nucleic acid single linear627 GCCGGCGGAC GUCAAGAA 18 54 base pairs nucleic acid single linear 628CGAGGCUGAG AAGCACGUAC CAGAGAAACA CACGUUGUGG UACAUUACCU GGUA 54 18 basepairs nucleic acid single linear 629 ACGUGCUGCU CAGCCUCG 18 54 basepairs nucleic acid single linear 630 GACCCCGAAG AAGAGCAGAC CAGAGAAACACACGUUGUGG UACAUUACCU GGUA 54 18 base pairs nucleic acid single linear631 CUGCUCAGCC UCGGGGUC 18 54 base pairs nucleic acid single linear 632CCUUGGCGAG AAGCGCGAAC CAGAGAAACA CACGUUGUGG UACAUUACCU GGUA 54 18 basepairs nucleic acid single linear 633 UCGCGCCGCU CGCCAAGG 18 54 basepairs nucleic acid single linear 634 GGCCUGCAAG AAGAACUCAC CAGAGAAACACACGUUGUGG UACAUUACCU GGUA 54 18 base pairs nucleic acid single linear635 GAGUUCGGCC UGCAGGCC 18 54 base pairs nucleic acid single linear 636CGCGCGAGAG AAGGGGGCAC CAGAGAAACA CACGUUGUGG UACAUUACCU GGUA 54 18 basepairs nucleic acid single linear 637 GCCCCCUGAU CUCGCGCG 18 54 basepairs nucleic acid single linear 638 AUAAACGAAG AAGCAUAUAC CAGAGAAACACACGUUGUGG UACAUUACCU GGUA 54 18 base pairs nucleic acid single linear639 AUAUGCUGUU UCGUUUAU 18 54 base pairs nucleic acid single linear 640CACAAGCAAG AAGCUACAAC CAGAGAAACA CACGUUGUGG UACAUUACCU GGUA 54 18 basepairs nucleic acid single linear 641 UGUAGCUGCU UGCUUGUG 18 54 basepairs nucleic acid single linear 642 AAUUACCGAG AAGUACUUAC CAGAGAAACACACGUUGUGG UACAUUACCU GGUA 54 18 base pairs nucleic acid single linear643 AAGUACCGAU CGGUAAUU 18 17 base pairs nucleic acid single linear 644CGCGCCCUCU GCCGCUU 17 17 base pairs nucleic acid single linear 645GUCCAGGUUA CACAUUC 17 17 base pairs nucleic acid single linear 646CUGCCGCUUG UUCGUUC 17 17 base pairs nucleic acid single linear 647UCCAGGUUAC ACAUUCA 17 17 base pairs nucleic acid single linear 648CCGCUUGUUC GUUCCUC 17 17 base pairs nucleic acid single linear 649UUACACAUUC AAUGCCA 17 17 base pairs nucleic acid single linear 650CGCUUGUUCG UUCCUCG 17 17 base pairs nucleic acid single linear 651UACACAUUCA AUGCCAC 17 17 base pairs nucleic acid single linear 652UUGUUCGUUC CUCGCGC 17 17 base pairs nucleic acid single linear 653UGCCACCUCA CAAGAUU 17 17 base pairs nucleic acid single linear 654UGUUCGUUCC UCGCGCU 17 17 base pairs nucleic acid single linear 655CACAAGAUUG AAAUUUU 17 17 base pairs nucleic acid single linear 656UCGUUCCUCG CGCUCGC 17 17 base pairs nucleic acid single linear 657AUUGAAAUUU UCAAGUC 17 17 base pairs nucleic acid single linear 658CUCGCGCUCG CCACCAG 17 17 base pairs nucleic acid single linear 659UUGAAAUUUU CAAGUCG 17 17 base pairs nucleic acid single linear 660ACACACAUCC CAAUCUC 17 17 base pairs nucleic acid single linear 661UGAAAUUUUC AAGUCGC 17 17 base pairs nucleic acid single linear 662AUCCCAAUCU CGCGAGG 17 17 base pairs nucleic acid single linear 663GAAAUUUUCA AGUCGCU 17 17 base pairs nucleic acid single linear 664CCCAAUCUCG CGAGGGC 17 17 base pairs nucleic acid single linear 665UUUCAAGUCG CUUGAUG 17 17 base pairs nucleic acid single linear 666AGCAGGGUCU GCGGCGG 17 17 base pairs nucleic acid single linear 667AAGUCGCUUG AUGAUUG 17 17 base pairs nucleic acid single linear 668GCCGCGCUUC CGGCUCC 17 17 base pairs nucleic acid single linear 669UUGAUGAUUG GGCUAGA 17 17 base pairs nucleic acid single linear 670CCGCGCUUCC GGCUCCC 17 17 base pairs nucleic acid single linear 671AUUGGGCUAG AGAUAAU 17 17 base pairs nucleic acid single linear 672UUCCGGCUCC CCUUCCC 17 17 base pairs nucleic acid single linear 673CUAGAGAUAA UAUCUUG 17 17 base pairs nucleic acid single linear 674GCUCCCCUUC CCAUUGG 17 17 base pairs nucleic acid single linear 675GAGAUAAUAU CUUGACG 17 17 base pairs nucleic acid single linear 676CUCCCCUUCC CAUUGGC 17 17 base pairs nucleic acid single linear 677GAUAAUAUCU UGACGCA 17 17 base pairs nucleic acid single linear 678CUUCCCAUUG GCCUCCA 17 17 base pairs nucleic acid single linear 679UAAUAUCUUG ACGCAUC 17 17 base pairs nucleic acid single linear 680AUUGGCCUCC ACGAUGG 17 17 base pairs nucleic acid single linear 681UGACGCAUCU CAAGCCA 17 17 base pairs nucleic acid single linear 682AUGGCGCUCC GCCUCAA 17 17 base pairs nucleic acid single linear 683ACGCAUCUCA AGCCAGU 17 17 base pairs nucleic acid single linear 684CUCCGCCUCA ACGACGU 17 17 base pairs nucleic acid single linear 685AAGCCAGUCG AGAAGUG 17 17 base pairs nucleic acid single linear 686AACGACGUCG CGCUCUG 17 17 base pairs nucleic acid single linear 687AGAAGUGUUG GCAGCCA 17 17 base pairs nucleic acid single linear 688GUCGCGCUCU GCCUCUC 17 17 base pairs nucleic acid single linear 689CACAGGAUUU CCUCCCG 17 17 base pairs nucleic acid single linear 690CUCUGCCUCU CCCCGCC 17 17 base pairs nucleic acid single linear 691ACAGGAUUUC CUCCCGG 17 17 base pairs nucleic acid single linear 692CUGCCUCUCC CCGCCGC 17 17 base pairs nucleic acid single linear 693CAGGAUUUCC UCCCGGA 17 17 base pairs nucleic acid single linear 694CCGCCGCUCG CCGCCCG 17 17 base pairs nucleic acid single linear 695GAUUUCCUCC CGGACCC 17 17 base pairs nucleic acid single linear 696CGGCAGGUUC GUCGCCG 17 17 base pairs nucleic acid single linear 697CCCAGCAUCU GAAGGAU 17 17 base pairs nucleic acid single linear 698GGCAGGUUCG UCGCCGU 17 17 base pairs nucleic acid single linear 699UGAAGGAUUU CAUGAUG 17 17 base pairs nucleic acid single linear 700AGGUUCGUCG CCGUCGC 17 17 base pairs nucleic acid single linear 701GAAGGAUUUC AUGAUGA 17 17 base pairs nucleic acid single linear 702GUCGCCGUCG CCUCCAU 17 17 base pairs nucleic acid single linear 703AAGGAUUUCA UGAUGAA 17 17 base pairs nucleic acid single linear 704CGUCGCCUCC AUGACGU 17 17 base pairs nucleic acid single linear 705GAUGAAGUUA AGGAGCU 17 17 base pairs nucleic acid single linear 706CAUGACGUCC GCCGUCU 17 17 base pairs nucleic acid single linear 707AUGAAGUUAA GGAGCUC 17 17 base pairs nucleic acid single linear 708UCCGCCGUCU CCACCAA 17 17 base pairs nucleic acid single linear 709AAGGAGCUCA GAGAACG 17 17 base pairs nucleic acid single linear 710CGCCGUCUCC ACCAAGG 17 17 base pairs nucleic acid single linear 711AAGGAAAUCC CUGAUGA 17 17 base pairs nucleic acid single linear 712ACCAAGGUCG AGAAUAA 17 17 base pairs nucleic acid single linear 713CUGAUGAUUA UUUUGUU 17 17 base pairs nucleic acid single linear 714UCGAGAAUAA GAAGCCA 17 17 base pairs nucleic acid single linear 715UGAUGAUUAU UUUGUUU 17 17 base pairs nucleic acid single linear 716GAAGCCAUUU GCUCCUC 17 17 base pairs nucleic acid single linear 717AUGAUUAUUU UGUUUGU 17 17 base pairs nucleic acid single linear 718AAGCCAUUUG CUCCUCC 17 17 base pairs nucleic acid single linear 719UGAUUAUUUU GUUUGUU 17 17 base pairs nucleic acid single linear 720CAUUUGCUCC UCCAAGG 17 17 base pairs nucleic acid single linear 721GAUUAUUUUG UUUGUUU 17 17 base pairs nucleic acid single linear 722UUGCUCCUCC AAGGGAG 17 17 base pairs nucleic acid single linear 723UAUUUUGUUU GUUUGGU 17 17 base pairs nucleic acid single linear 724AGGGAGGUAC AUGUCCA 17 17 base pairs nucleic acid single linear 725AUUUUGUUUG UUUGGUG 17 17 base pairs nucleic acid single linear 726GUACAUGUCC AGGUUAC 17 17 base pairs nucleic acid single linear 727UUGUUUGUUU GGUGGGA 17 17 base pairs nucleic acid single linear 728UGUUUGUUUG GUGGGAG 17 17 base pairs nucleic acid single linear 729ACACUGCUCG UCACGCC 17 17 base pairs nucleic acid single linear 730GACAUGAUUA CCGAGGA 17 17 base pairs nucleic acid single linear 731CUGCUCGUCA CGCCAAG 17 17 base pairs nucleic acid single linear 732ACAUGAUUAC CGAGGAA 17 17 base pairs nucleic acid single linear 733CAAGGACUUU GGCGACU 17 17 base pairs nucleic acid single linear 734AGGAAGCUCU ACCAACA 17 17 base pairs nucleic acid single linear 735AAGGACUUUG GCGACUU 17 17 base pairs nucleic acid single linear 736GAAGCUCUAC CAACAUA 17 17 base pairs nucleic acid single linear 737UGGCGACUUA AAGCUUG 17 17 base pairs nucleic acid single linear 738ACCAACAUAC CAGACUA 17 17 base pairs nucleic acid single linear 739GGCGACUUAA AGCUUGC 17 17 base pairs nucleic acid single linear 740ACCAGACUAU GCUUAAC 17 17 base pairs nucleic acid single linear 741UUAAAGCUUG CACAAAU 17 17 base pairs nucleic acid single linear 742ACUAUGCUUA ACACCCU 17 17 base pairs nucleic acid single linear 743GCACAAAUCU GCGGCAU 17 17 base pairs nucleic acid single linear 744CUAUGCUUAA CACCCUC 17 17 base pairs nucleic acid single linear 745UGCGGCAUCA UCGCCUC 17 17 base pairs nucleic acid single linear 746AACACCCUCG ACGGUGU 17 17 base pairs nucleic acid single linear 747GGCAUCAUCG CCUCAGA 17 17 base pairs nucleic acid single linear 748GACGGUGUCA GAGAUGA 17 17 base pairs nucleic acid single linear 749CAUCGCCUCA GAUGAGA 17 17 base pairs nucleic acid single linear 750UGGGCUGUUU GGACGAG 17 17 base pairs nucleic acid single linear 751AACUGCGUAC ACCAAGA 17 17 base pairs nucleic acid single linear 752GGGCUGUUUG GACGAGG 17 17 base pairs nucleic acid single linear 753ACCAAGAUCG UGGAGAA 17 17 base pairs nucleic acid single linear 754AUGGUGAUCU GCUCAAC 17 17 base pairs nucleic acid single linear 755GAAGCUGUUU GAGAUCG 17 17 base pairs nucleic acid single linear 756GAUCUGCUCA ACAAGUA 17 17 base pairs nucleic acid single linear 757AAGCUGUUUG AGAUCGA 17 17 base pairs nucleic acid single linear 758CAACAAGUAU AUGUACC 17 17 base pairs nucleic acid single linear 759UUUGAGAUCG ACCCUGA 17 17 base pairs nucleic acid single linear 760ACAAGUAUAU GUACCUC 17 17 base pairs nucleic acid single linear 761CUGAUGGUAC CGUGGUC 17 17 base pairs nucleic acid single linear 762GUAUAUGUAC CUCACUG 17 17 base pairs nucleic acid single linear 763ACCGUGGUCG CUCUGGC 17 17 base pairs nucleic acid single linear 764AUGUACCUCA CUGGGAG 17 17 base pairs nucleic acid single linear 765UGGUCGCUCU GGCUGAC 17 17 base pairs nucleic acid single linear 766GGGUGGAUAU GAGGCAG 17 17 base pairs nucleic acid single linear 767AAGAAGAUCU CAAUGCC 17 17 base pairs nucleic acid single linear 768AGGCAGAUUG AGAAGAC 17 17 base pairs nucleic acid single linear 769GAAGAUCUCA AUGCCUG 17 17 base pairs nucleic acid single linear 770AAGACAAUUC AGUAUCU 17 17 base pairs nucleic acid single linear 771CCUGAUGUUU GACGGGC 17 17 base pairs nucleic acid single linear 772AGACAAUUCA GUAUCUU 17 17 base pairs nucleic acid single linear 773CUGAUGUUUG ACGGGCA 17 17 base pairs nucleic acid single linear 774AAUUCAGUAU CUUAUUG 17 17 base pairs nucleic acid single linear 775CAAGCUGUUC GAGCACU 17 17 base pairs nucleic acid single linear 776UUCAGUAUCU UAUUGGC 17 17 base pairs nucleic acid single linear 777AAGCUGUUCG AGCACUU 17 17 base pairs nucleic acid single linear 778CAGUAUCUUA UUGGCUC 17 17 base pairs nucleic acid single linear 779CGAGCACUUC UCCAUGG 17 17 base pairs nucleic acid single linear 780AGUAUCUUAU UGGCUCU 17 17 base pairs nucleic acid single linear 781GAGCACUUCU CCAUGGU 17 17 base pairs nucleic acid single linear 782UAUCUUAUUG GCUCUGG 17 17 base pairs nucleic acid single linear 783GCACUUCUCC AUGGUCG 17 17 base pairs nucleic acid single linear 784UAUUGGCUCU GGAAUGG 17 17 base pairs nucleic acid single linear 785UCCAUGGUCG CGCAGAG 17 17 base pairs nucleic acid single linear 786GAAUGGAUCC UAGGACU 17 17 base pairs nucleic acid single linear 787CAGAGGCUUG GCGUUUA 17 17 base pairs nucleic acid single linear 788UGGAUCCUAG GACUGAG 17 17 base pairs nucleic acid single linear 789CUUGGCGUUU ACACCGC 17 17 base pairs nucleic acid single linear 790CUGAGAAUAA UCCUUAU 17 17 base pairs nucleic acid single linear 791UUGGCGUUUA CACCGCC 17 17 base pairs nucleic acid single linear 792AGAAUAAUCC UUAUCUU 17 17 base pairs nucleic acid single linear 793UGGCGUUUAC ACCGCCA 17 17 base pairs nucleic acid single linear 794AUAAUCCUUA UCUUGGU 17 17 base pairs nucleic acid single linear 795CAGGGACUAC GCCGACA 17 17 base pairs nucleic acid single linear 796UAAUCCUUAU CUUGGUU 17 17 base pairs nucleic acid single linear 797GCCGACAUCC UCGAGUU 17 17 base pairs nucleic acid single linear 798AUCCUUAUCU UGGUUUC 17 17 base pairs nucleic acid single linear 799GACAUCCUCG AGUUCCU 17 17 base pairs nucleic acid single linear 800CCUUAUCUUG GUUUCAU 17 17 base pairs nucleic acid single linear 801CCUCGAGUUC CUCGUCG 17 17 base pairs nucleic acid single linear 802AUCUUGGUUU CAUCUAC 17 17 base pairs nucleic acid single linear 803CUCGAGUUCC UCGUCGA 17 17 base pairs nucleic acid single linear 804UCUUGGUUUC AUCUACA 17 17 base pairs nucleic acid single linear 805GAGUUCCUCG UCGACAG 17 17 base pairs nucleic acid single linear 806CUUGGUUUCA UCUACAC 17 17 base pairs nucleic acid single linear 807UUCCUCGUCG ACAGGUG 17 17 base pairs nucleic acid single linear 808GGUUUCAUCU ACACCUC 17 17 base pairs nucleic acid single linear 809UGACUGGUCU GUCGGGU 17 17 base pairs nucleic acid single linear 810UUUCAUCUAC ACCUCCU 17 17 base pairs nucleic acid single linear 811UGGUCUGUCG GGUGAAG 17 17 base pairs nucleic acid single linear 812CUACACCUCC UUCCAAG 17 17 base pairs nucleic acid single linear 813GCAGGACUAC CUUUGCA 17 17 base pairs nucleic acid single linear 814CACCUCCUUC CAAGAGC 17 17 base pairs nucleic acid single linear 815GACUACCUUU GCACCCU 17 17 base pairs nucleic acid single linear 816ACCUCCUUCC AAGAGCG 17 17 base pairs nucleic acid single linear 817ACUACCUUUG CACCCUU 17 17 base pairs nucleic acid single linear 818GGCGACCUUC AUCUCAC 17 17 base pairs nucleic acid single linear 819UGCACCCUUG CUUCAAG 17 17 base pairs nucleic acid single linear 820GCGACCUUCA UCUCACA 17 17 base pairs nucleic acid single linear 821CCCUUGCUUC AAGAAUC 17 17 base pairs nucleic acid single linear 822ACCUUCAUCU CACACGG 17 17 base pairs nucleic acid single linear 823CCUUGCUUCA AGAAUCA 17 17 base pairs nucleic acid single linear 824CUUCAUCUCA CACGGGA 17 17 base pairs nucleic acid single linear 825UCAAGAAUCA GGAGGCU 17 17 base pairs nucleic acid single linear 826CGCUGCCUUU CAGCUGG 17 17 base pairs nucleic acid single linear 827UUUGAUGUAC AACCUGU 17 17 base pairs nucleic acid single linear 828GCUGCCUUUC AGCUGGG 17 17 base pairs nucleic acid single linear 829CAUGCCGUAC UUUGUCU 17 17 base pairs nucleic acid single linear 830CUGCCUUUCA GCUGGGU 17 17 base pairs nucleic acid single linear 831GCCGUACUUU GUCUGUC 17 17 base pairs nucleic acid single linear 832AGCUGGGUAU ACGGUAG 17 17 base pairs nucleic acid single linear 833CCGUACUUUG UCUGUCG 17 17 base pairs nucleic acid single linear 834CUGGGUAUAC GGUAGGG 17 17 base pairs nucleic acid single linear 835UACUUUGUCU GUCGCUG 17 17 base pairs nucleic acid single linear 836UAUACGGUAG GGACGUC 17 17 base pairs nucleic acid single linear 837UUGUCUGUCG CUGGCGG 17 17 base pairs nucleic acid single linear 838AGGGACGUCC AACUGUG 17 17 base pairs nucleic acid single linear 839CGGUGUGUUU CGGUAUG 17 17 base pairs nucleic acid single linear 840UGUGAGAUCG GAAACCU 17 17 base pairs nucleic acid single linear 841GGUGUGUUUC GGUAUGU 17 17 base pairs nucleic acid single linear 842GCUGCGGUCU GCUUAGA 17 17 base pairs nucleic acid single linear 843GUGUGUUUCG GUAUGUU 17 17 base pairs nucleic acid single linear 844GGUCUGCUUA GACAAGA 17 17 base pairs nucleic acid single linear 845GUUUCGGUAU GUUAUUU 17 17 base pairs nucleic acid single linear 846GUCUGCUUAG ACAAGAC 17 17 base pairs nucleic acid single linear 847CGGUAUGUUA UUUGAGU 17 17 base pairs nucleic acid single linear 848UGCUGUGUCU GCGUUAC 17 17 base pairs nucleic acid single linear 849GGUAUGUUAU UUGAGUU 17 17 base pairs nucleic acid single linear 850GUCUGCGUUA CAUAGGU 17 17 base pairs nucleic acid single linear 851UAUGUUAUUU GAGUUGC 17 17 base pairs nucleic acid single linear 852UCUGCGUUAC AUAGGUC 17 17 base pairs nucleic acid single linear 853AUGUUAUUUG AGUUGCU 17 17 base pairs nucleic acid single linear 854CGUUACAUAG GUCUCCA 17 17 base pairs nucleic acid single linear 855AUUUGAGUUG CUCAGAU 17 17 base pairs nucleic acid single linear 856ACAUAGGUCU CCAGGUU 17 17 base pairs nucleic acid single linear 857GAGUUGCUCA GAUCUGU 17 17 base pairs nucleic acid single linear 858AUAGGUCUCC AGGUUUU 17 17 base pairs nucleic acid single linear 859GCUCAGAUCU GUUAAAA 17 17 base pairs nucleic acid single linear 860CUCCAGGUUU UGAUCAA 17 17 base pairs nucleic acid single linear 861AGAUCUGUUA AAAAAAA 17 17 base pairs nucleic acid single linear 862UCCAGGUUUU GAUCAAA 17 17 base pairs nucleic acid single linear 863GAUCUGUUAA AAAAAAA 17 17 base pairs nucleic acid single linear 864CCAGGUUUUG AUCAAAU 17 17 base pairs nucleic acid single linear 865GUUUUGAUCA AAUGGUC 17 17 base pairs nucleic acid single linear 866CAAAUGGUCC CGUGUCG 17 17 base pairs nucleic acid single linear 867UCCCGUGUCG UCUUAUA 17 17 base pairs nucleic acid single linear 868CGUGUCGUCU UAUAGAG 17 17 base pairs nucleic acid single linear 869UGUCGUCUUA UAGAGCG 17 17 base pairs nucleic acid single linear 870GUCGUCUUAU AGAGCGA 17 17 base pairs nucleic acid single linear 871CGUCUUAUAG AGCGAUA 17 17 base pairs nucleic acid single linear 872AGAGCGAUAG GAGAACG 17 17 base pairs nucleic acid single linear 873GAACGUGUUG GUCUGUG 17 17 base pairs nucleic acid single linear 874GUGUUGGUCU GUGGUGU 17 17 base pairs nucleic acid single linear 875UGUGGUGUAG CUUUGUU 17 17 base pairs nucleic acid single linear 876GUGUAGCUUU GUUUUUA 17 17 base pairs nucleic acid single linear 877UGUAGCUUUG UUUUUAU 17 17 base pairs nucleic acid single linear 878AGCUUUGUUU UUAUUUU 17 17 base pairs nucleic acid single linear 879GCUUUGUUUU UAUUUUG 17 17 base pairs nucleic acid single linear 880CUUUGUUUUU AUUUUGU 17 17 base pairs nucleic acid single linear 881UUUGUUUUUA UUUUGUA 17 17 base pairs nucleic acid single linear 882UUGUUUUUAU UUUGUAU 17 17 base pairs nucleic acid single linear 883GUUUUUAUUU UGUAUUU 17 17 base pairs nucleic acid single linear 884UUUUUAUUUU GUAUUUU 17 17 base pairs nucleic acid single linear 885UUUUAUUUUG UAUUUUU 17 17 base pairs nucleic acid single linear 886UAUUUUGUAU UUUUCUG 17 17 base pairs nucleic acid single linear 887UUUUGUAUUU UUCUGCU 17 17 base pairs nucleic acid single linear 888UUUGUAUUUU UCUGCUU 17 17 base pairs nucleic acid single linear 889UUGUAUUUUU CUGCUUU 17 17 base pairs nucleic acid single linear 890UGUAUUUUUC UGCUUUG 17 17 base pairs nucleic acid single linear 891GUAUUUUUCU GCUUUGA 17 17 base pairs nucleic acid single linear 892UUUCUGCUUU GAUGUAC 17 17 base pairs nucleic acid single linear 893UUCUGCUUUG AUGUACA 17 27 base pairs nucleic acid single linear Theletter “N” stands for any base. 894 AAGCGGCACU GAUGANGAAA GGGCGCG 27 27base pairs nucleic acid single linear The letter “N” stands for anybase. 895 GAACGAACCU GAUGANGAAA GCGGCAG 27 27 base pairs nucleic acidsingle linear The letter “N” stands for any base. 896 GAGGAACGCUGAUGANGAAA CAAGCGG 27 27 base pairs nucleic acid single linear Theletter “N” stands for any base. 897 CGAGGAACCU GAUGANGAAA ACAAGCG 27 27base pairs nucleic acid single linear The letter “N” stands for anybase. 898 GCGCGAGGCU GAUGANGAAA CGAACAA 27 27 base pairs nucleic acidsingle linear The letter “N” stands for any base. 899 AGCGCGAGCUGAUGANGAAA ACGAACA 27 27 base pairs nucleic acid single linear Theletter “N” stands for any base. 900 GCGAGCGCCU GAUGANGAAA GGAACGA 27 27base pairs nucleic acid single linear The letter “N” stands for anybase. 901 CUGGUGGCCU GAUGANGAAA GCGCGAG 27 27 base pairs nucleic acidsingle linear The letter “N” stands for any base. 902 GAGAUUGGCUGAUGANGAAA UGUGUGU 27 27 base pairs nucleic acid single linear Theletter “N” stands for any base. 903 CCUCGCGACU GAUGANGAAA UUGGGAU 27 27base pairs nucleic acid single linear The letter “N” stands for anybase. 904 GCCCUCGCCU GAUGANGAAA GAUUGGG 27 27 base pairs nucleic acidsingle linear The letter “N” stands for any base. 905 CCGCCGCACUGAUGANGAAA CCCUGCU 27 27 base pairs nucleic acid single linear Theletter “N” stands for any base. 906 GGAGCCGGCU GAUGANGAAA GCGCGGC 27 27base pairs nucleic acid single linear The letter “N” stands for anybase. 907 GGGAGCCGCU GAUGANGAAA AGCGCGG 27 27 base pairs nucleic acidsingle linear The letter “N” stands for any base. 908 GGGAAGGGCUGAUGANGAAA GCCGGAA 27 27 base pairs nucleic acid single linear Theletter “N” stands for any base. 909 CCAAUGGGCU GAUGANGAAA GGGGAGC 27 27base pairs nucleic acid single linear The letter “N” stands for anybase. 910 GCCAAUGGCU GAUGANGAAA AGGGGAG 27 27 base pairs nucleic acidsingle linear The letter “N” stands for any base. 911 UGGAGGCCCUGAUGANGAAA UGGGAAG 27 27 base pairs nucleic acid single linear Theletter “N” stands for any base. 912 CCAUCGUGCU GAUGANGAAA GGCCAAU 27 27base pairs nucleic acid single linear The letter “N” stands for anybase. 913 UUGAGGCGCU GAUGANGAAA GCGCCAU 27 27 base pairs nucleic acidsingle linear The letter “N” stands for any base. 914 ACGUCGUUCUGAUGANGAAA GGCGGAG 27 27 base pairs nucleic acid single linear Theletter “N” stands for any base. 915 CAGAGCGCCU GAUGANGAAA CGUCGUU 27 27base pairs nucleic acid single linear The letter “N” stands for anybase. 916 GAGAGGCACU GAUGANGAAA GCGCGAC 27 27 base pairs nucleic acidsingle linear The letter “N” stands for any base. 917 GGCGGGGACUGAUGANGAAA GGCAGAG 27 27 base pairs nucleic acid single linear Theletter “N” stands for any base. 918 GCGGCGGGCU GAUGANGAAA GAGGCAG 27 27base pairs nucleic acid single linear The letter “N” stands for anybase. 919 CGGGCGGCCU GAUGANGAAA GCGGCGG 27 27 base pairs nucleic acidsingle linear The letter “N” stands for any base. 920 CGGCGACGCUGAUGANGAAA CCUGCCG 27 27 base pairs nucleic acid single linear Theletter “N” stands for any base. 921 ACGGCGACCU GAUGANGAAA ACCUGCC 27 27base pairs nucleic acid single linear The letter “N” stands for anybase. 922 GCGACGGCCU GAUGANGAAA CGAACCU 27 27 base pairs nucleic acidsingle linear The letter “N” stands for any base. 923 AUGGAGGCCUGAUGANGAAA CGGCGAC 27 27 base pairs nucleic acid single linear Theletter “N” stands for any base. 924 ACGUCAUGCU GAUGANGAAA GGCGACG 27 27base pairs nucleic acid single linear The letter “N” stands for anybase. 925 AGACGGCGCU GAUGANGAAA CGUCAUG 27 27 base pairs nucleic acidsingle linear The letter “N” stands for any base. 926 UUGGUGGACUGAUGANGAAA CGGCGGA 27 27 base pairs nucleic acid single linear Theletter “N” stands for any base. 927 CCUUGGUGCU GAUGANGAAA GACGGCG 27 27base pairs nucleic acid single linear The letter “N” stands for anybase. 928 UUAUUCUCCU GAUGANGAAA CCUUGGU 27 27 base pairs nucleic acidsingle linear The letter “N” stands for any base. 929 UGGCUUCUCUGAUGANGAAA UUCUCGA 27 27 base pairs nucleic acid single linear Theletter “N” stands for any base. 930 GAGGAGCACU GAUGANGAAA UGGCUUC 27 27base pairs nucleic acid single linear The letter “N” stands for anybase. 931 GGAGGAGCCU GAUGANGAAA AUGGCUU 27 27 base pairs nucleic acidsingle linear The letter “N” stands for any base. 932 CCUUGGAGCUGAUGANGAAA GCAAAUG 27 27 base pairs nucleic acid single linear Theletter “N” stands for any base. 933 CUCCCUUGCU GAUGANGAAA GGAGCAA 27 27base pairs nucleic acid single linear The letter “N” stands for anybase. 934 UGGACAUGCU GAUGANGAAA CCUCCCU 27 27 base pairs nucleic acidsingle linear The letter “N” stands for any base. 935 GUAACCUGCUGAUGANGAAA CAUGUAC 27 27 base pairs nucleic acid single linear Theletter “N” stands for any base. 936 GAAUGUGUCU GAUGANGAAA CCUGGAC 27 27base pairs nucleic acid single linear The letter “N” stands for anybase. 937 UGAAUGUGCU GAUGANGAAA ACCUGGA 27 27 base pairs nucleic acidsingle linear The letter “N” stands for any base. 938 UGGCAUUGCUGAUGANGAAA UGUGUAA 27 27 base pairs nucleic acid single linear Theletter “N” stands for any base. 939 GUGGCAUUCU GAUGANGAAA AUGUGUA 27 27base pairs nucleic acid single linear The letter “N” stands for anybase. 940 AAUCUUGUCU GAUGANGAAA GGUGGCA 27 27 base pairs nucleic acidsingle linear The letter “N” stands for any base. 941 AAAAUUUCCUGAUGANGAAA UCUUGUG 27 27 base pairs nucleic acid single linear Theletter “N” stands for any base. 942 GACUUGAACU GAUGANGAAA UUUCAAU 27 27base pairs nucleic acid single linear The letter “N” stands for anybase. 943 CGACUUGACU GAUGANGAAA AUUUCAA 27 27 base pairs nucleic acidsingle linear The letter “N” stands for any base. 944 GCGACUUGCUGAUGANGAAA AAUUUCA 27 27 base pairs nucleic acid single linear Theletter “N” stands for any base. 945 AGCGACUUCU GAUGANGAAA AAAUUUC 27 27base pairs nucleic acid single linear The letter “N” stands for anybase. 946 CAUCAAGCCU GAUGANGAAA CUUGAAA 27 27 base pairs nucleic acidsingle linear The letter “N” stands for any base. 947 CAAUCAUCCUGAUGANGAAA GCGACUU 27 27 base pairs nucleic acid single linear Theletter “N” stands for any base. 948 UCUAGCCCCU GAUGANGAAA UCAUCAA 27 27base pairs nucleic acid single linear The letter “N” stands for anybase. 949 AUUAUCUCCU GAUGANGAAA GCCCAAU 27 27 base pairs nucleic acidsingle linear The letter “N” stands for any base. 950 CAAGAUAUCUGAUGANGAAA UCUCUAG 27 27 base pairs nucleic acid single linear Theletter “N” stands for any base. 951 CGUCAAGACU GAUGANGAAA UUAUCUC 27 27base pairs nucleic acid single linear The letter “N” stands for anybase. 952 UGCGUCAACU GAUGANGAAA UAUUAUC 27 27 base pairs nucleic acidsingle linear The letter “N” stands for any base. 953 GAUGCGUCCUGAUGANGAAA GAUAUUA 27 27 base pairs nucleic acid single linear Theletter “N” stands for any base. 954 UGGCUUGACU GAUGANGAAA UGCGUCA 27 27base pairs nucleic acid single linear The letter “N” stands for anybase. 955 ACUGGCUUCU GAUGANGAAA GAUGCGU 27 27 base pairs nucleic acidsingle linear The letter “N” stands for any base. 956 CACUUCUCCUGAUGANGAAA CUGGCUU 27 27 base pairs nucleic acid single linear Theletter “N” stands for any base. 957 UGGCUGCCCU GAUGANGAAA CACUUCU 27 27base pairs nucleic acid single linear The letter “N” stands for anybase. 958 CGGGAGGACU GAUGANGAAA UCCUGUG 27 27 base pairs nucleic acidsingle linear The letter “N” stands for any base. 959 CCGGGAGGCUGAUGANGAAA AUCCUGU 27 27 base pairs nucleic acid single linear Theletter “N” stands for any base. 960 UCCGGGAGCU GAUGANGAAA AAUCCUG 27 27base pairs nucleic acid single linear The letter “N” stands for anybase. 961 GGGUCCGGCU GAUGANGAAA GGAAAUC 27 27 base pairs nucleic acidsingle linear The letter “N” stands for any base. 962 AUCCUUCACUGAUGANGAAA UGCUGGG 27 27 base pairs nucleic acid single linear Theletter “N” stands for any base. 963 CAUCAUGACU GAUGANGAAA UCCUUCA 27 27base pairs nucleic acid single linear The letter “N” stands for anybase. 964 UCAUCAUGCU GAUGANGAAA AUCCUUC 27 27 base pairs nucleic acidsingle linear The letter “N” stands for any base. 965 UUCAUCAUCUGAUGANGAAA AAUCCUU 27 27 base pairs nucleic acid single linear Theletter “N” stands for any base. 966 AGCUCCUUCU GAUGANGAAA CUUCAUC 27 27base pairs nucleic acid single linear The letter “N” stands for anybase. 967 GAGCUCCUCU GAUGANGAAA ACUUCAU 27 27 base pairs nucleic acidsingle linear The letter “N” stands for any base. 968 CGUUCUCUCUGAUGANGAAA GCUCCUU 27 27 base pairs nucleic acid single linear Theletter “N” stands for any base. 969 UCAUCAGGCU GAUGANGAAA UUUCCUU 27 27base pairs nucleic acid single linear The letter “N” stands for anybase. 970 AACAAAAUCU GAUGANGAAA UCAUCAG 27 27 base pairs nucleic acidsingle linear The letter “N” stands for any base. 971 AAACAAAACUGAUGANGAAA AUCAUCA 27 27 base pairs nucleic acid single linear Theletter “N” stands for any base. 972 ACAAACAACU GAUGANGAAA UAAUCAU 27 27base pairs nucleic acid single linear The letter “N” stands for anybase. 973 AACAAACACU GAUGANGAAA AUAAUCA 27 27 base pairs nucleic acidsingle linear The letter “N” stands for any base. 974 AAACAAACCUGAUGANGAAA AAUAAUC 27 27 base pairs nucleic acid single linear Theletter “N” stands for any base. 975 ACCAAACACU GAUGANGAAA CAAAAUA 27 27base pairs nucleic acid single linear The letter “N” stands for anybase. 976 CACCAAACCU GAUGANGAAA ACAAAAU 27 27 base pairs nucleic acidsingle linear The letter “N” stands for any base. 977 UCCCACCACUGAUGANGAAA CAAACAA 27 27 base pairs nucleic acid single linear Theletter “N” stands for any base. 978 CUCCCACCCU GAUGANGAAA ACAAACA 27 27base pairs nucleic acid single linear The letter “N” stands for anybase. 979 UCCUCGGUCU GAUGANGAAA UCAUGUC 27 27 base pairs nucleic acidsingle linear The letter “N” stands for any base. 980 UUCCUCGGCUGAUGANGAAA AUCAUGU 27 27 base pairs nucleic acid single linear Theletter “N” stands for any base. 981 UGUUGGUACU GAUGANGAAA GCUUCCU 27 27base pairs nucleic acid single linear The letter “N” stands for anybase. 982 UAUGUUGGCU GAUGANGAAA GAGCUUC 27 27 base pairs nucleic acidsingle linear The letter “N” stands for any base. 983 UAGUCUGGCUGAUGANGAAA UGUUGGU 27 27 base pairs nucleic acid single linear Theletter “N” stands for any base. 984 GUUAAGCACU GAUGANGAAA GUCUGGU 27 27base pairs nucleic acid single linear The letter “N” stands for anybase. 985 AGGGUGUUCU GAUGANGAAA GCAUAGU 27 27 base pairs nucleic acidsingle linear The letter “N” stands for any base. 986 GAGGGUGUCUGAUGANGAAA AGCAUAG 27 27 base pairs nucleic acid single linear Theletter “N” stands for any base. 987 ACACCGUCCU GAUGANGAAA GGGUGUU 27 27base pairs nucleic acid single linear The letter “N” stands for anybase. 988 UCAUCUCUCU GAUGANGAAA CACCGUC 27 27 base pairs nucleic acidsingle linear The letter “N” stands for any base. 989 CUCGUCCACUGAUGANGAAA CAGCCCA 27 27 base pairs nucleic acid single linear Theletter “N” stands for any base. 990 CCUCGUCCCU GAUGANGAAA ACAGCCC 27 27base pairs nucleic acid single linear The letter “N” stands for anybase. 991 GUUGAGCACU GAUGANGAAA UCACCAU 27 27 base pairs nucleic acidsingle linear The letter “N” stands for any base. 992 UACUUGUUCUGAUGANGAAA GCAGAUC 27 27 base pairs nucleic acid single linear Theletter “N” stands for any base. 993 GGUACAUACU GAUGANGAAA CUUGUUG 27 27base pairs nucleic acid single linear The letter “N” stands for anybase. 994 GAGGUACACU GAUGANGAAA UACUUGU 27 27 base pairs nucleic acidsingle linear The letter “N” stands for any base. 995 CAGUGAGGCUGAUGANGAAA CAUAUAC 27 27 base pairs nucleic acid single linear Theletter “N” stands for any base. 996 CUCCCAGUCU GAUGANGAAA GGUACAU 27 27base pairs nucleic acid single linear The letter “N” stands for anybase. 997 CUGCCUCACU GAUGANGAAA UCCACCC 27 27 base pairs nucleic acidsingle linear The letter “N” stands for any base. 998 GUCUUCUCCUGAUGANGAAA UCUGCCU 27 27 base pairs nucleic acid single linear Theletter “N” stands for any base. 999 AGAUACUGCU GAUGANGAAA UUGUCUU 27 27base pairs nucleic acid single linear The letter “N” stands for anybase. 1000 AAGAUACUCU GAUGANGAAA AUUGUCU 27 27 base pairs nucleic acidsingle linear The letter “N” stands for any base. 1001 CAAUAAGACUGAUGANGAAA CUGAAUU 27 27 base pairs nucleic acid single linear Theletter “N” stands for any base. 1002 GCCAAUAACU GAUGANGAAA UACUGAA 27 27base pairs nucleic acid single linear The letter “N” stands for anybase. 1003 GAGCCAAUCU GAUGANGAAA GAUACUG 27 27 base pairs nucleic acidsingle linear The letter “N” stands for any base. 1004 AGAGCCAACUGAUGANGAAA AGAUACU 27 27 base pairs nucleic acid single linear Theletter “N” stands for any base. 1005 CCAGAGCCCU GAUGANGAAA UAAGAUA 27 27base pairs nucleic acid single linear The letter “N” stands for anybase. 1006 CCAUUCCACU GAUGANGAAA GCCAAUA 27 27 base pairs nucleic acidsingle linear The letter “N” stands for any base. 1007 AGUCCUAGCUGAUGANGAAA UCCAUUC 27 27 base pairs nucleic acid single linear Theletter “N” stands for any base. 1008 CUCAGUCCCU GAUGANGAAA GGAUCCA 27 27base pairs nucleic acid single linear The letter “N” stands for anybase. 1009 AUAAGGAUCU GAUGANGAAA UUCUCAG 27 27 base pairs nucleic acidsingle linear The letter “N” stands for any base. 1010 AAGAUAAGCUGAUGANGAAA UUAUUCU 27 27 base pairs nucleic acid single linear Theletter “N” stands for any base. 1011 ACCAAGAUCU GAUGANGAAA GGAUUAU 27 27base pairs nucleic acid single linear The letter “N” stands for anybase. 1012 AACCAAGACU GAUGANGAAA AGGAUUA 27 27 base pairs nucleic acidsingle linear The letter “N” stands for any base. 1013 GAAACCAACUGAUGANGAAA UAAGGAU 27 27 base pairs nucleic acid single linear Theletter “N” stands for any base. 1014 AUGAAACCCU GAUGANGAAA GAUAAGG 27 27base pairs nucleic acid single linear The letter “N” stands for anybase. 1015 GUAGAUGACU GAUGANGAAA CCAAGAU 27 27 base pairs nucleic acidsingle linear The letter “N” stands for any base. 1016 UGUAGAUGCUGAUGANGAAA ACCAAGA 27 27 base pairs nucleic acid single linear Theletter “N” stands for any base. 1017 GUGUAGAUCU GAUGANGAAA AACCAAG 27 27base pairs nucleic acid single linear The letter “N” stands for anybase. 1018 GAGGUGUACU GAUGANGAAA UGAAACC 27 27 base pairs nucleic acidsingle linear The letter “N” stands for any base. 1019 AGGAGGUGCUGAUGANGAAA GAUGAAA 27 27 base pairs nucleic acid single linear Theletter “N” stands for any base. 1020 CUUGGAAGCU GAUGANGAAA GGUGUAG 27 27base pairs nucleic acid single linear The letter “N” stands for anybase. 1021 GCUCUUGGCU GAUGANGAAA GGAGGUG 27 27 base pairs nucleic acidsingle linear The letter “N” stands for any base. 1022 CGCUCUUGCUGAUGANGAAA AGGAGGU 27 27 base pairs nucleic acid single linear Theletter “N” stands for any base. 1023 GUGAGAUGCU GAUGANGAAA GGUCGCC 27 27base pairs nucleic acid single linear The letter “N” stands for anybase. 1024 UGUGAGAUCU GAUGANGAAA AGGUCGC 27 27 base pairs nucleic acidsingle linear The letter “N” stands for any base. 1025 CCGUGUGACUGAUGANGAAA UGAAGGU 27 27 base pairs nucleic acid single linear Theletter “N” stands for any base. 1026 UCCCGUGUCU GAUGANGAAA GAUGAAG 27 27base pairs nucleic acid single linear The letter “N” stands for anybase. 1027 GGCGUGACCU GAUGANGAAA GCAGUGU 27 27 base pairs nucleic acidsingle linear The letter “N” stands for any base. 1028 CUUGGCGUCUGAUGANGAAA CGAGCAG 27 27 base pairs nucleic acid single linear Theletter “N” stands for any base. 1029 AGUCGCCACU GAUGANGAAA GUCCUUG 27 27base pairs nucleic acid single linear The letter “N” stands for anybase. 1030 AAGUCGCCCU GAUGANGAAA AGUCCUU 27 27 base pairs nucleic acidsingle linear The letter “N” stands for any base. 1031 CAAGCUUUCUGAUGANGAAA GUCGCCA 27 27 base pairs nucleic acid single linear Theletter “N” stands for any base. 1032 GCAAGCUUCU GAUGANGAAA AGUCGCC 27 27base pairs nucleic acid single linear The letter “N” stands for anybase. 1033 AUUUGUGCCU GAUGANGAAA GCUUUAA 27 27 base pairs nucleic acidsingle linear The letter “N” stands for any base. 1034 AUGCCGCACUGAUGANGAAA UUUGUGC 27 27 base pairs nucleic acid single linear Theletter “N” stands for any base. 1035 GAGGCGAUCU GAUGANGAAA UGCCGCA 27 27base pairs nucleic acid single linear The letter “N” stands for anybase. 1036 UCUGAGGCCU GAUGANGAAA UGAUGCC 27 27 base pairs nucleic acidsingle linear The letter “N” stands for any base. 1037 UCUCAUCUCUGAUGANGAAA GGCGAUG 27 27 base pairs nucleic acid single linear Theletter “N” stands for any base. 1038 UCUUGGUGCU GAUGANGAAA CGCAGUU 27 27base pairs nucleic acid single linear The letter “N” stands for anybase. 1039 UUCUCCACCU GAUGANGAAA UCUUGGU 27 27 base pairs nucleic acidsingle linear The letter “N” stands for any base. 1040 CGAUCUCACUGAUGANGAAA CAGCUUC 27 27 base pairs nucleic acid single linear Theletter “N” stands for any base. 1041 UCGAUCUCCU GAUGANGAAA ACAGCUU 27 27base pairs nucleic acid single linear The letter “N” stands for anybase. 1042 UCAGGGUCCU GAUGANGAAA UCUCAAA 27 27 base pairs nucleic acidsingle linear The letter “N” stands for any base. 1043 GACCACGGCUGAUGANGAAA CCAUCAG 27 27 base pairs nucleic acid single linear Theletter “N” stands for any base. 1044 GCCAGAGCCU GAUGANGAAA CCACGGU 27 27base pairs nucleic acid single linear The letter “N” stands for anybase. 1045 GUCAGCCACU GAUGANGAAA GCGACCA 27 27 base pairs nucleic acidsingle linear The letter “N” stands for any base. 1046 GGCAUUGACUGAUGANGAAA UCUUCUU 27 27 base pairs nucleic acid single linear Theletter “N” stands for any base. 1047 CAGGCAUUCU GAUGANGAAA GAUCUUC 27 27base pairs nucleic acid single linear The letter “N” stands for anybase. 1048 GCCCGUCACU GAUGANGAAA CAUCAGG 27 27 base pairs nucleic acidsingle linear The letter “N” stands for any base. 1049 UGCCCGUCCUGAUGANGAAA ACAUCAG 27 27 base pairs nucleic acid single linear Theletter “N” stands for any base. 1050 AGUGCUCGCU GAUGANGAAA CAGCUUG 27 27base pairs nucleic acid single linear The letter “N” stands for anybase. 1051 AAGUGCUCCU GAUGANGAAA ACAGCUU 27 27 base pairs nucleic acidsingle linear The letter “N” stands for any base. 1052 CCAUGGAGCUGAUGANGAAA GUGCUCG 27 27 base pairs nucleic acid single linear Theletter “N” stands for any base. 1053 ACCAUGGACU GAUGANGAAA AGUGCUC 27 27base pairs nucleic acid single linear The letter “N” stands for anybase. 1054 CGACCAUGCU GAUGANGAAA GAAGUGC 27 27 base pairs nucleic acidsingle linear The letter “N” stands for any base. 1055 CUCUGCGCCUGAUGANGAAA CCAUGGA 27 27 base pairs nucleic acid single linear Theletter “N” stands for any base. 1056 UAAACGCCCU GAUGANGAAA GCCUCUG 27 27base pairs nucleic acid single linear The letter “N” stands for anybase. 1057 GCGGUGUACU GAUGANGAAA CGCCAAG 27 27 base pairs nucleic acidsingle linear The letter “N” stands for any base. 1058 GGCGGUGUCUGAUGANGAAA ACGCCAA 27 27 base pairs nucleic acid single linear Theletter “N” stands for any base. 1059 UGGCGGUGCU GAUGANGAAA AACGCCA 27 27base pairs nucleic acid single linear The letter “N” stands for anybase. 1060 UGUCGGCGCU GAUGANGAAA GUCCCUG 27 27 base pairs nucleic acidsingle linear The letter “N” stands for any base. 1061 AACUCGAGCUGAUGANGAAA UGUCGGC 27 27 base pairs nucleic acid single linear Theletter “N” stands for any base. 1062 AGGAACUCCU GAUGANGAAA GGAUGUC 27 27base pairs nucleic acid single linear The letter “N” stands for anybase. 1063 CGACGAGGCU GAUGANGAAA CUCGAGG 27 27 base pairs nucleic acidsingle linear The letter “N” stands for any base. 1064 UCGACGAGCUGAUGANGAAA ACUCGAG 27 27 base pairs nucleic acid single linear Theletter “N” stands for any base. 1065 CUGUCGACCU GAUGANGAAA GGAACUC 27 27base pairs nucleic acid single linear The letter “N” stands for anybase. 1066 CACCUGUCCU GAUGANGAAA CGAGGAA 27 27 base pairs nucleic acidsingle linear The letter “N” stands for any base. 1067 ACCCGACACUGAUGANGAAA CCAGUCA 27 27 base pairs nucleic acid single linear Theletter “N” stands for any base. 1068 CUUCACCCCU GAUGANGAAA CAGACCA 27 27base pairs nucleic acid single linear The letter “N” stands for anybase. 1069 UGCAAAGGCU GAUGANGAAA GUCCUGC 27 27 base pairs nucleic acidsingle linear The letter “N” stands for any base. 1070 AGGGUGCACUGAUGANGAAA GGUAGUC 27 27 base pairs nucleic acid single linear Theletter “N” stands for any base. 1071 AAGGGUGCCU GAUGANGAAA AGGUAGU 27 27base pairs nucleic acid single linear The letter “N” stands for anybase. 1072 CUUGAAGCCU GAUGANGAAA GGGUGCA 27 27 base pairs nucleic acidsingle linear The letter “N” stands for any base. 1073 GAUUCUUGCUGAUGANGAAA GCAAGGG 27 27 base pairs nucleic acid single linear Theletter “N” stands for any base. 1074 UGAUUCUUCU GAUGANGAAA AGCAAGG 27 27base pairs nucleic acid single linear The letter “N” stands for anybase. 1075 AGCCUCCUCU GAUGANGAAA UUCUUGA 27 27 base pairs nucleic acidsingle linear The letter “N” stands for any base. 1076 CCAGCUGACUGAUGANGAAA GGCAGCG 27 27 base pairs nucleic acid single linear Theletter “N” stands for any base. 1077 CCCAGCUGCU GAUGANGAAA AGGCAGC 27 27base pairs nucleic acid single linear The letter “N” stands for anybase. 1078 ACCCAGCUCU GAUGANGAAA AAGGCAG 27 27 base pairs nucleic acidsingle linear The letter “N” stands for any base. 1079 CUACCGUACUGAUGANGAAA CCCAGCU 27 27 base pairs nucleic acid single linear Theletter “N” stands for any base. 1080 CCCUACCGCU GAUGANGAAA UACCCAG 27 27base pairs nucleic acid single linear The letter “N” stands for anybase. 1081 GACGUCCCCU GAUGANGAAA CCGUAUA 27 27 base pairs nucleic acidsingle linear The letter “N” stands for any base. 1082 CACAGUUGCUGAUGANGAAA CGUCCCU 27 27 base pairs nucleic acid single linear Theletter “N” stands for any base. 1083 AGGUUUCCCU GAUGANGAAA UCUCACA 27 27base pairs nucleic acid single linear The letter “N” stands for anybase. 1084 UCUAAGCACU GAUGANGAAA CCGCAGC 27 27 base pairs nucleic acidsingle linear The letter “N” stands for any base. 1085 UCUUGUCUCUGAUGANGAAA GCAGACC 27 27 base pairs nucleic acid single linear Theletter “N” stands for any base. 1086 GUCUUGUCCU GAUGANGAAA AGCAGAC 27 27base pairs nucleic acid single linear The letter “N” stands for anybase. 1087 GUAACGCACU GAUGANGAAA CACAGCA 27 27 base pairs nucleic acidsingle linear The letter “N” stands for any base. 1088 ACCUAUGUCUGAUGANGAAA CGCAGAC 27 27 base pairs nucleic acid single linear Theletter “N” stands for any base. 1089 GACCUAUGCU GAUGANGAAA ACGCAGA 27 27base pairs nucleic acid single linear The letter “N” stands for anybase. 1090 UGGAGACCCU GAUGANGAAA UGUAACG 27 27 base pairs nucleic acidsingle linear The letter “N” stands for any base. 1091 AACCUGGACUGAUGANGAAA CCUAUGU 27 27 base pairs nucleic acid single linear Theletter “N” stands for any base. 1092 AAAACCUGCU GAUGANGAAA GACCUAU 27 27base pairs nucleic acid single linear The letter “N” stands for anybase. 1093 UUGAUCAACU GAUGANGAAA CCUGGAG 27 27 base pairs nucleic acidsingle linear The letter “N” stands for any base. 1094 UUUGAUCACUGAUGANGAAA ACCUGGA 27 27 base pairs nucleic acid single linear Theletter “N” stands for any base. 1095 AUUUGAUCCU GAUGANGAAA AACCUGG 27 27base pairs nucleic acid single linear The letter “N” stands for anybase. 1096 GACCAUUUCU GAUGANGAAA UCAAAAC 27 27 base pairs nucleic acidsingle linear The letter “N” stands for any base. 1097 CGACACGGCUGAUGANGAAA CCAUUUG 27 27 base pairs nucleic acid single linear Theletter “N” stands for any base. 1098 UAUAAGACCU GAUGANGAAA CACGGGA 27 27base pairs nucleic acid single linear The letter “N” stands for anybase. 1099 CUCUAUAACU GAUGANGAAA CGACACG 27 27 base pairs nucleic acidsingle linear The letter “N” stands for any base. 1100 CGCUCUAUCUGAUGANGAAA GACGACA 27 27 base pairs nucleic acid single linear Theletter “N” stands for any base. 1101 UCGCUCUACU GAUGANGAAA AGACGAC 27 27base pairs nucleic acid single linear The letter “N” stands for anybase. 1102 UAUCGCUCCU GAUGANGAAA UAAGACG 27 27 base pairs nucleic acidsingle linear The letter “N” stands for any base. 1103 CGUUCUCCCUGAUGANGAAA UCGCUCU 27 27 base pairs nucleic acid single linear Theletter “N” stands for any base. 1104 CACAGACCCU GAUGANGAAA CACGUUC 27 27base pairs nucleic acid single linear The letter “N” stands for anybase. 1105 ACACCACACU GAUGANGAAA CCAACAC 27 27 base pairs nucleic acidsingle linear The letter “N” stands for any base. 1106 AACAAAGCCUGAUGANGAAA CACCACA 27 27 base pairs nucleic acid single linear Theletter “N” stands for any base. 1107 UAAAAACACU GAUGANGAAA GCUACAC 27 27base pairs nucleic acid single linear The letter “N” stands for anybase. 1108 AUAAAAACCU GAUGANGAAA AGCUACA 27 27 base pairs nucleic acidsingle linear The letter “N” stands for any base. 1109 AAAAUAAACUGAUGANGAAA CAAAGCU 27 27 base pairs nucleic acid single linear Theletter “N” stands for any base. 1110 CAAAAUAACU GAUGANGAAA ACAAAGC 27 27base pairs nucleic acid single linear The letter “N” stands for anybase. 1111 ACAAAAUACU GAUGANGAAA AACAAAG 27 27 base pairs nucleic acidsingle linear The letter “N” stands for any base. 1112 UACAAAAUCUGAUGANGAAA AAACAAA 27 27 base pairs nucleic acid single linear Theletter “N” stands for any base. 1113 AUACAAAACU GAUGANGAAA AAAACAA 27 27base pairs nucleic acid single linear The letter “N” stands for anybase. 1114 AAAUACAACU GAUGANGAAA UAAAAAC 27 27 base pairs nucleic acidsingle linear The letter “N” stands for any base. 1115 AAAAUACACUGAUGANGAAA AUAAAAA 27 27 base pairs nucleic acid single linear Theletter “N” stands for any base. 1116 AAAAAUACCU GAUGANGAAA AAUAAAA 27 27base pairs nucleic acid single linear The letter “N” stands for anybase. 1117 CAGAAAAACU GAUGANGAAA CAAAAUA 27 27 base pairs nucleic acidsingle linear The letter “N” stands for any base. 1118 AGCAGAAACUGAUGANGAAA UACAAAA 27 27 base pairs nucleic acid single linear Theletter “N” stands for any base. 1119 AAGCAGAACU GAUGANGAAA AUACAAA 27 27base pairs nucleic acid single linear The letter “N” stands for anybase. 1120 AAAGCAGACU GAUGANGAAA AAUACAA 27 27 base pairs nucleic acidsingle linear The letter “N” stands for any base. 1121 CAAAGCAGCUGAUGANGAAA AAAUACA 27 27 base pairs nucleic acid single linear Theletter “N” stands for any base. 1122 UCAAAGCACU GAUGANGAAA AAAAUAC 27 27base pairs nucleic acid single linear The letter “N” stands for anybase. 1123 GUACAUCACU GAUGANGAAA GCAGAAA 27 27 base pairs nucleic acidsingle linear The letter “N” stands for any base. 1124 UGUACAUCCUGAUGANGAAA AGCAGAA 27 27 base pairs nucleic acid single linear Theletter “N” stands for any base. 1125 ACAGGUUGCU GAUGANGAAA CAUCAAA 27 27base pairs nucleic acid single linear The letter “N” stands for anybase. 1126 AGACAAAGCU GAUGANGAAA CGGCAUG 27 27 base pairs nucleic acidsingle linear The letter “N” stands for any base. 1127 GACAGACACUGAUGANGAAA GUACGGC 27 27 base pairs nucleic acid single linear Theletter “N” stands for any base. 1128 CGACAGACCU GAUGANGAAA AGUACGG 27 27base pairs nucleic acid single linear The letter “N” stands for anybase. 1129 CAGCGACACU GAUGANGAAA CAAAGUA 27 27 base pairs nucleic acidsingle linear The letter “N” stands for any base. 1130 CCGCCAGCCUGAUGANGAAA CAGACAA 27 27 base pairs nucleic acid single linear Theletter “N” stands for any base. 1131 CAUACCGACU GAUGANGAAA CACACCG 27 27base pairs nucleic acid single linear The letter “N” stands for anybase. 1132 ACAUACCGCU GAUGANGAAA ACACACC 27 27 base pairs nucleic acidsingle linear The letter “N” stands for any base. 1133 AACAUACCCUGAUGANGAAA AACACAC 27 27 base pairs nucleic acid single linear Theletter “N” stands for any base. 1134 AAAUAACACU GAUGANGAAA CCGAAAC 27 27base pairs nucleic acid single linear The letter “N” stands for anybase. 1135 ACUCAAAUCU GAUGANGAAA CAUACCG 27 27 base pairs nucleic acidsingle linear The letter “N” stands for any base. 1136 AACUCAAACUGAUGANGAAA ACAUACC 27 27 base pairs nucleic acid single linear Theletter “N” stands for any base. 1137 GCAACUCACU GAUGANGAAA UAACAUA 27 27base pairs nucleic acid single linear The letter “N” stands for anybase. 1138 AGCAACUCCU GAUGANGAAA AUAACAU 27 27 base pairs nucleic acidsingle linear The letter “N” stands for any base. 1139 AUCUGAGCCUGAUGANGAAA CUCAAAU 27 27 base pairs nucleic acid single linear Theletter “N” stands for any base. 1140 ACAGAUCUCU GAUGANGAAA GCAACUC 27 27base pairs nucleic acid single linear The letter “N” stands for anybase. 1141 UUUUAACACU GAUGANGAAA UCUGAGC 27 27 base pairs nucleic acidsingle linear The letter “N” stands for any base. 1142 UUUUUUUUCUGAUGANGAAA CAGAUCU 27 27 base pairs nucleic acid single linear Theletter “N” stands for any base. 1143 UUUUUUUUCU GAUGANGAAA ACAGAUC 27 54base pairs nucleic acid single linear 1144 GAACAAGCAG AAGAGGGCACCAGAGAAACA CACGUUGUGG UACAUUACCU GGUA 54 18 base pairs nucleic acidsingle linear 1145 GCCCUCUGCC GCUUGUUC 18 54 base pairs nucleic acidsingle linear 1146 AACGAACAAG AAGCAGAGAC CAGAGAAACA CACGUUGUGGUACAUUACCU GGUA 54 18 base pairs nucleic acid single linear 1147CUCUGCCGCU UGUUCGUU 18 54 base pairs nucleic acid single linear 1148GGAAGCGCAG AAGCCGCCAC CAGAGAAACA CACGUUGUGG UACAUUACCU GGUA 54 18 basepairs nucleic acid single linear 1149 GGCGGCGGCC GCGCUUCC 18 54 basepairs nucleic acid single linear 1150 GGAAGGGGAG AAGGAAGCAC CAGAGAAACACACGUUGUGG UACAUUACCU GGUA 54 18 base pairs nucleic acid single linear1151 GCUUCCGGCU CCCCUUCC 18 54 base pairs nucleic acid single linear1152 GUCGUUGAAG AAGAGCGCAC CAGAGAAACA CACGUUGUGG UACAUUACCU GGUA 54 18base pairs nucleic acid single linear 1153 GCGCUCCGCC UCAACGAC 18 54base pairs nucleic acid single linear 1154 CGGGGAGAAG AAGAGCGCACCAGAGAAACA CACGUUGUGG UACAUUACCU GGUA 54 18 base pairs nucleic acidsingle linear 1155 GCGCUCUGCC UCUCCCCG 18 54 base pairs nucleic acidsingle linear 1156 CGGCGAGCAG AAGGGAGAAC CAGAGAAACA CACGUUGUGGUACAUUACCU GGUA 54 18 base pairs nucleic acid single linear 1157UCUCCCCGCC GCUCGCCG 18 54 base pairs nucleic acid single linear 1158GGGCGGCGAG AAGCGGGGAC CAGAGAAACA CACGUUGUGG UACAUUACCU GGUA 54 18 basepairs nucleic acid single linear 1159 CCCCGCCGCU CGCCGCCC 18 54 basepairs nucleic acid single linear 1160 CGGCGGCGAG AAGCGAGCAC CAGAGAAACACACGUUGUGG UACAUUACCU GGUA 54 18 base pairs nucleic acid single linear1161 GCUCGCCGCC CGCCGCCG 18 54 base pairs nucleic acid single linear1162 GCGGCGGCAG AAGGCGGCAC CAGAGAAACA CACGUUGUGG UACAUUACCU GGUA 54 18base pairs nucleic acid single linear 1163 GCCGCCCGCC GCCGCCGC 18 54base pairs nucleic acid single linear 1164 GCGGCGGCAG AAGCGGGCACCAGAGAAACA CACGUUGUGG UACAUUACCU GGUA 54 18 base pairs nucleic acidsingle linear 1165 GCCCGCCGCC GCCGCCGC 18 54 base pairs nucleic acidsingle linear 1166 GCUGCGGCAG AAGCGGCGAC CAGAGAAACA CACGUUGUGGUACAUUACCU GGUA 54 18 base pairs nucleic acid single linear 1167CGCCGCCGCC GCCGCAGC 18 54 base pairs nucleic acid single linear 1168GCUGCUGCAG AAGCGGCGAC CAGAGAAACA CACGUUGUGG UACAUUACCU GGUA 54 18 basepairs nucleic acid single linear 1169 CGCCGCCGCC GCAGCAGC 18 54 basepairs nucleic acid single linear 1170 AUGGAGGCAG AAGCGACGAC CAGAGAAACACACGUUGUGG UACAUUACCU GGUA 54 18 base pairs nucleic acid single linear1171 CGUCGCCGUC GCCUCCAU 18 54 base pairs nucleic acid single linear1172 GUGGAGACAG AAGACGUCAC CAGAGAAACA CACGUUGUGG UACAUUACCU GGUA 54 18base pairs nucleic acid single linear 1173 GACGUCCGCC GUCUCCAC 18 54base pairs nucleic acid single linear 1174 UUGGUGGAAG AAGCGGACACCAGAGAAACA CACGUUGUGG UACAUUACCU GGUA 54 18 base pairs nucleic acidsingle linear 1175 GUCCGCCGUC UCCACCAA 18 54 base pairs nucleic acidsingle linear 1176 CACUUCUCAG AAGGCUUGAC CAGAGAAACA CACGUUGUGGUACAUUACCU GGUA 54 18 base pairs nucleic acid single linear 1177CAAGCCAGUC GAGAAGUG 18 54 base pairs nucleic acid single linear 1178GAUGCUGGAG AAGGGAGGAC CAGAGAAACA CACGUUGUGG UACAUUACCU GGUA 54 18 basepairs nucleic acid single linear 1179 CCUCCCGGAC CCAGCAUC 18 54 basepairs nucleic acid single linear 1180 AAAUAAUCAG AAGGGAUUAC CAGAGAAACACACGUUGUGG UACAUUACCU GGUA 54 18 base pairs nucleic acid single linear1181 AAUCCCUGAU GAUUAUUU 18 54 base pairs nucleic acid single linear1182 UAAGCAUAAG AAGGUAUGAC CAGAGAAACA CACGUUGUGG UACAUUACCU GGUA 54 18base pairs nucleic acid single linear 1183 CAUACCAGAC UAUGCUUA 18 54base pairs nucleic acid single linear 1184 ACAGCCCAAG AAGUGGGGACCAGAGAAACA CACGUUGUGG UACAUUACCU GGUA 54 18 base pairs nucleic acidsingle linear 1185 CCCCACUGCC UGGGCUGU 18 54 base pairs nucleic acidsingle linear 1186 CUCGUCCAAG AAGCCCAGAC CAGAGAAACA CACGUUGUGGUACAUUACCU GGUA 54 18 base pairs nucleic acid single linear 1187CUGGGCUGUU UGGACGAG 18 54 base pairs nucleic acid single linear 1188UUCUCCUCAG AAGUCCAUAC CAGAGAAACA CACGUUGUGG UACAUUACCU GGUA 54 18 basepairs nucleic acid single linear 1189 AUGGACUGCU GAGGAGAA 18 54 basepairs nucleic acid single linear 1190 ACUUGUUGAG AAGAUCACAC CAGAGAAACACACGUUGUGG UACAUUACCU GGUA 54 18 base pairs nucleic acid single linear1191 GUGAUCUGCU CAACAAGU 18 54 base pairs nucleic acid single linear1192 UCUUCUCAAG AAGCCUCAAC CAGAGAAACA CACGUUGUGG UACAUUACCU GGUA 54 18base pairs nucleic acid single linear 1193 UGAGGCAGAU UGAGAAGA 18 54base pairs nucleic acid single linear 1194 GCGUGACGAG AAGUGUUCACCAGAGAAACA CACGUUGUGG UACAUUACCU GGUA 54 18 base pairs nucleic acidsingle linear 1195 GAACACUGCU CGUCACGC 18 54 base pairs nucleic acidsingle linear 1196 CGCUUCUCAG AAGAGGCGAC CAGAGAAACA CACGUUGUGGUACAUUACCU GGUA 54 18 base pairs nucleic acid single linear 1197CGCCUCAGAU GAGAAGCG 18 54 base pairs nucleic acid single linear 1198CGAUCUCAAG AAGCUUCUAC CAGAGAAACA CACGUUGUGG UACAUUACCU GGUA 54 18 basepairs nucleic acid single linear 1199 AGAAGCUGUU UGAGAUCG 18 54 basepairs nucleic acid single linear 1200 ACGGUACCAG AAGGGUCGAC CAGAGAAACACACGUUGUGG UACAUUACCU GGUA 54 18 base pairs nucleic acid single linear1201 CGACCCUGAU GGUACCGU 18 54 base pairs nucleic acid single linear1202 AUCAGGUGAG AAGGCAUUAC CAGAGAAACA CACGUUGUGG UACAUUACCU GGUA 54 18base pairs nucleic acid single linear 1203 AAUGCCUGCC CACCUGAU 18 54base pairs nucleic acid single linear 1204 CGUCAAACAG AAGGUGGGACCAGAGAAACA CACGUUGUGG UACAUUACCU GGUA 54 18 base pairs nucleic acidsingle linear 1205 CCCACCUGAU GUUUGACG 18 54 base pairs nucleic acidsingle linear 1206 AGUGCUCGAG AAGCUUGUAC CAGAGAAACA CACGUUGUGGUACAUUACCU GGUA 54 18 base pairs nucleic acid single linear 1207ACAAGCUGUU CGAGCACU 18 54 base pairs nucleic acid single linear 1208ACAGACCAAG AAGGCUCGAC CAGAGAAACA CACGUUGUGG UACAUUACCU GGUA 54 18 basepairs nucleic acid single linear 1209 CGAGCCUGAC UGGUCUGU 18 54 basepairs nucleic acid single linear 1210 CUUCACCCAG AAGACCAGAC CAGAGAAACACACGUUGUGG UACAUUACCU GGUA 54 18 base pairs nucleic acid single linear1211 CUGGUCUGUC GGGUGAAG 18 54 base pairs nucleic acid single linear1212 AGCUGAAAAG AAGCGUGCAC CAGAGAAACA CACGUUGUGG UACAUUACCU GGUA 54 18base pairs nucleic acid single linear 1213 GCACGCUGCC UUUCAGCU 18 54base pairs nucleic acid single linear 1214 GUAUACCCAG AAGAAAGGACCAGAGAAACA CACGUUGUGG UACAUUACCU GGUA 54 18 base pairs nucleic acidsingle linear 1215 CCUUUCAGCU GGGUAUAC 18 54 base pairs nucleic acidsingle linear 1216 CAGACCGCAG AAGGUUUCAC CAGAGAAACA CACGUUGUGGUACAUUACCU GGUA 54 18 base pairs nucleic acid single linear 1217GAAACCUGCU GCGGUCUG 18 54 base pairs nucleic acid single linear 1218UCUAAGCAAG AAGCAGCAAC CAGAGAAACA CACGUUGUGG UACAUUACCU GGUA 54 18 basepairs nucleic acid single linear 1219 UGCUGCGGUC UGCUUAGA 18 54 basepairs nucleic acid single linear 1220 CUUGUCUAAG AAGACCGCAC CAGAGAAACACACGUUGUGG UACAUUACCU GGUA 54 18 base pairs nucleic acid single linear1221 GCGGUCUGCU UAGACAAG 18 54 base pairs nucleic acid single linear1222 GCAGACACAG AAGGUCUUAC CAGAGAAACA CACGUUGUGG UACAUUACCU GGUA 54 18base pairs nucleic acid single linear 1223 AAGACCUGCU GUGUCUGC 18 54base pairs nucleic acid single linear 1224 UACAUCAAAG AAGAAAAAACCAGAGAAACA CACGUUGUGG UACAUUACCU GGUA 54 18 base pairs nucleic acidsingle linear 1225 UUUUUCUGCU UUGAUGUA 18 54 base pairs nucleic acidsingle linear 1226 CCGCCAGCAG AAGACAAAAC CAGAGAAACA CACGUUGUGGUACAUUACCU GGUA 54 18 base pairs nucleic acid single linear 1227UUUGUCUGUC GCUGGCGG 18 54 base pairs nucleic acid single linear 1228UUUAACAGAG AAGAGCAAAC CAGAGAAACA CACGUUGUGG UACAUUACCU GGUA 54 18 basepairs nucleic acid single linear 1229 UUGCUCAGAU CUGUUAAA 18 11 basepairs nucleic acid single linear The letter “N” stands for any base. Theletter “H” stands for A, U or C. 1230 NNNNUHNNNN N 11 28 base pairsnucleic acid single linear The letter “N” stands for any base. 1231NNNNNCUGAN GAGNNNNNNC GAAANNNN 28 15 base pairs nucleic acid singlelinear The letter “N” stands for any base. The leter “Y” stands for U orC. The letter “H” stands for A, U or C. 1232 NNNNNNNYNG HYNNN 15 47 basepairs nucleic acid single linear The letter “N” stands for any base.1233 NNNNGAAGNN NNNNNNNNNA AAHANNNNNN NACAUUACNN NNNNNNN 47 49 basepairs nucleic acid single linear The letter “N” stands for any base.1234 CUCCACCUCC UCGCGGUNNN NNNNGGGCUA CUUCGGUAGG CUAAGGGAG 49 176 basepairs nucleic acid single linear 1235 GGGAAAGCUU GCGAAGGGCG UCGUCGCCCCGAGCGGUAGU AAGCAGGGAA CUCACCUCCA 60 AUUUCAGUAC UGAAAUUGUC GUAGCAGUUGACUACUGUUA UGUGAUUGGU AGAGGCUAAG 120 UGACGGUAUU GGCGUAAGUC AGUAUUGCAGCACAGCACAA GCCCGCUUGC GAGAAU 176 91 base pairs nucleic acid doublelinear 1236 AAGCTTGCAT GCCTGCAGGC CGGCCTTAAT TAAGCGGCCG CGTTTAAACGCCCGGGCATT 60 TTCGAACGTA CGGACGTCCG GCCGGAATTA ATTCGCCGGC GCAAATTTGCGGGCCCGTAA TAAATGGCGC GCCGCGATCG CTTGCAGATC T 91 ATTTACCGCG CGGCGCTAGCGAACGTCTAG A 10 base pairs nucleic acid single linear 1237 GGCGAAAGCC 10109 base pairs nucleic acid single linear 1238 CGCGGATCCT GGTAGGACTGATGAGGCCGA AAGGCCGAAA TGTTGTGCTG ATGAGGCCGA 60 AAGGCCGAAA TGCAGAAAGCGGTCTTTGCG TCCCTGTAGA TGCCGTGGC 109 106 base pairs nucleic acid singlelinear 1239 CGCGAGCTCG GCCCTCTCTT TCGGCCTTTC GGCCTCATCA GGTGCTACCTCAAGAGCAAC 60 TACCAGTTTC GGCCTTTCGG CCTCATCAGC CACGGCATCT ACAGGG 106 47base pairs nucleic acid single linear 1240 GATCCGATGC CGTGGCTGATGAGGCCGAAA GGCCGAAACT GGTAGTT 47 43 base pairs nucleic acid singlelinear 1241 AACTACCAGT TTCGGCCTTT CGGCCTCATC AGCCACGGCA TCG 43 88 basepairs nucleic acid single linear 1242 CTGCAGGCCG GCCTTAATTA AGCGGCCGCGTTTAAACGCC CGGGCATTTA AATGGCGCGC 60 CGCGATCGCT TGCAGATCTG CATGGGTG 88 20base pairs nucleic acid single linear 1243 GGGGACTCTA GAGGATCCAG 20 10base pairs nucleic acid single linear 1244 GACGGATCTG 10 24 base pairsnucleic acid single linear 1245 TGAGATCTGA GCTCGAATTT CCCC 24 19 basepairs nucleic acid single linear 1246 CTGCAGATCT GCATGGGTG 19 13 basepairs nucleic acid single linear 1247 GGGGACTCTA GAG 13 16 base pairsnucleic acid single linear 1248 GACGGATCCG TCGACC 16 10 base pairsnucleic acid single linear 1249 GAATTTCCCC 10 25 base pairs nucleic acidsingle linear 1250 GATCCGCCCG GGGCCCGGGC GGTAC 25 17 base pairs nucleicacid single linear 1251 CGCCCGGGCC CCGGGCG 17 30 base pairs nucleic acidsingle linear 1252 GTGCCCACAA TGGCGCTCCG CCTCAACGAC 30 57 base pairsnucleic acid single linear 1253 TCATCACAGG TCCTCCTCGC TGATCAGCTTCTCCTCCAGT TGGACCTGCC TACCGTA 57 57 base pairs nucleic acid singlelinear 1254 TACGGTAGGG ACGTCCAACT GGAGGAGAAG CTGATCAGCG AGGAGGACCTGTGATGA 57 18 base pairs nucleic acid single linear 1255 CGCAAGACCGGCAACAGG 18 22 base pairs nucleic acid single linear 1256 TGGATTGATGTGATATCTCC AC 22 18 base pairs nucleic acid single linear 1257CGCAAGACCG GCAACAGG 18 31 base pairs nucleic acid single linear 1258CAGATCAAGT GCAAAGCTGC GGACGGATCT G 31 20 base pairs nucleic acid singlelinear 1259 ATCCGATGCC GTGGCTGATG 20 20 base pairs nucleic acid singlelinear 1260 GATGAGATCC GGTGGCATTG 20 20 base pairs nucleic acid singlelinear 1261 ATCCCCTTGG TGGACTGATG 20 31 base pairs nucleic acid singlelinear 1262 CAGATCAAGT GCAAAGCTGC GGACGGATCT G 31 6 amino acids aminoacid single linear peptide 1263 Ala Val Ala Ser Met Thr 1 5

What is claimed is:
 1. An isolated nucleic acid fragment comprising SEQID NO.
 1. 2. A maize plant transformed with a construct comprising inthe 5′ to 3′ direction of Transcription: a promoter functional in saidplant; a double strand DNA (dsDNA) comprising SEQ ID NO. 1, wherein thetranscript strand of said dsDNA is complementary to RNA endogenous tosaid plant; and a termination region functional in said plant.
 3. Atransgenic plant that is a progeny of the maize plant of claim
 2. 4. Anexpression vector comprising a nucleic acid sequence encoding at leastone nucleic acid of claim 1, in a manner which allows expression of saidnucleic acid.
 5. A plant cell comprising the expression vector of claim4.
 6. A maize plant transformed with the expression vector of claim 4.7. A transgenic plant that is a progeny of the maize plant of claim 6.8. The transgenic plant of claim 2, 3, 6, or 7, wherein the plant istransformed by Agrobacterium, electroporation, whiskers, or bybombardment with DNA coated microprojectiles.
 9. The transgenic plant ofclaim 8, wherein said bombardment with DNA coated microprojectiles isdone with a gene gun.
 10. The transgenic plant of claim 2, 3, 6, or 7,wherein the plant contains a selectable marker comprising the bar geneor a gene encoding resistance to a selection agent selected from thegroup consisting of chlorosulfuron, hygromycin, bromoxynil, andkanamycin.
 11. The transgenic plant of claim 2 or 3, wherein the doublestrand DNA is operably linked to a cauliflower mosaic virus (35S)promoter or a promoter from a gene encoding a protein selected from thegroup consisting of octopine synthetase, nopaline synthase, mannopinesynthetase, ribulose-1,6-bisphosphate (RUBP) carboxylase small subunit(ssu), beta-conglycinin, phaseolin, napin, gamma zein, globulin, ADH,heat shock protein, actin, and ubiquitin.